Philippa Kaur

1.Foster, S. A. & Endler, J. A. Geographic Variation in Behavior: Perspectives on Evolutionary Mechanisms 1–336 (Oxford University Press, 1999).Book 

Google Scholar 
2.Mundiger, P. C. Microgeographic and macrogeographic variation in the acquired vocalizations of birds. In Acoustic Communication in Birds 147–208 (Academic Press, 1982).
Google Scholar 
3.Green, S. Dialects in Japanese monkeys: Vocal learning and cultural transmission of locale-specific vocal behavior?. Z. Tierpsychol. J. Comp. Ethol. 38(3), 304–314 (1975).CAS 
Article 

Google Scholar 
4.Hodun, A., Snowdon, C. T. & Soini, P. Subspecific variation in the long calls of the tamarin, Saguinus fusckollis. Z. Tierpsychol. 57, 97–110 (1981).Article 

Google Scholar 
5.Ford, J. K. B. & Fisher, H. D. Group-specific dialects of killer whales (Orcinus orca) in British Columbia. In Communication and Behavior of Whales 129–161 (Westview Press for the American Association for the Advancement of Science, 1983).
Google Scholar 
6.Filatova, O. A. et al. Call diversity in the North Pacific killer whale populations: Implications for dialect evolution and population history. Anim. Behav. 83, 595–603 (2012).Article 

Google Scholar 
7.Rendell, L. E. & Whitehead, H. Vocal clans in sperm whales (Physeter macrocephalus). Proc. Biol. Sci. R. Soc. 270, 225–231 (2003).CAS 
Article 

Google Scholar 
8.Gero, S., Whitehead, H. & Rendell, L. Individual, unit and vocal clan level identity cues in sperm whale codas. R. Soc. Open Sci. 3, 1–12 (2016).
Google Scholar 
9.Cise, A. M., Van Mahaffy, S. D., Baird, R. W., Mooney, T. A. & Barlow, J. Song of my people: Dialect differences among sympatric social groups of short-finned pilot whales in Hawai’i. Behav. Ecol. Sociobiol. 72, 1–13 (2018).Article 

Google Scholar 
10.Podos, J. & Warren, P. S. The evolution of geographic variation in birdsong. Adv. Study Behav. 37, 403–458 (2007).Article 

Google Scholar 
11.Walker, T. J. Factors responsible for intraspecific variation in the calling songs of crickets. Evolution 16, 407–428 (1962).Article 

Google Scholar 
12.Velásquez, N. A. Geographic variation in acoustic communication in anurans and its neuroethological implications. J. Physiol. 108, 167–173 (2014).
Google Scholar 
13.Amorim, T. O. S., Andriolo, A., Reis, S. S. & dos Santos, M. E. Vocalizations of Amazon river dolphins (Inia geoffrensis): Characterization, effect of physical environment and differences between populations. J. Acoust. Soc. Am. 139, 1285–1293 (2016).ADS 
PubMed 
Article 

Google Scholar 
14.Moron, J. R. et al. Spinner dolphin whistle in the Southwest Atlantic Ocean: Is there a geographic variation?. J. Acoust. Soc. Am. 138, 2495–2498 (2015).ADS 
PubMed 
Article 

Google Scholar 
15.Bjørgesæter, A., Ugland, K. I. & Bjørge, A. Geographic variation and acoustic structure of the underwater vocalization of harbor seal (Phoca vitulina) in Norway, Sweden and Scotland. J. Acoust. Soc. Am. 116, 2459–2468 (2004).ADS 
PubMed 
Article 

Google Scholar 
16.Janik, V. & Slater, P. The different roles of social learning in vocal communication. Anim. Behav. 60, 1–11 (2000).CAS 
PubMed 
Article 

Google Scholar 
17.Lameira, A. R., Delgado, R. A. & Wich, S. A. Review of geographic variation in terrestrial mammalian acoustic signals: Human speech variation in a comparative perspective. J. Evol. Psychol. 8, 309–332 (2010).Article 

Google Scholar 
18.Janik, V. Acoustic communication networks in marine mammals. In Animal Communication Networks 390–415 (University Press, 2005).
Google Scholar 
19.Deecke, V. B., Ford, J. K. B. & Spong, P. Dialect change in resident killer whales: Implications for vocal learning and cultural transmission. Anim. Behav. 60, 629–638 (2000).CAS 
PubMed 
Article 

Google Scholar 
20.Weilgart, L. & Whitehead, H. Group-specific dialects and geographical variation in coda repertoire in South Pacific sperm whales. Behav. Ecol. Sociobiol. 40, 277–285 (1997).Article 

Google Scholar 
21.Azevedo, A. F. & Van Sluys, M. Whistles of tucuxi dolphins (Sotalia fluviatilis) in Brazil: Comparisons among populations. J. Acoust. Soc. Am. 117, 1456–1464 (2005).ADS 
PubMed 
Article 

Google Scholar 
22.Bazúa-Durán, C. & Au, W. W. L. Geographic variations in the whistles of spinner dolphins (Stenella longirostris) of the Main Hawaiian Islands. J. Acoust. Soc. Am. 116, 3757–3769 (2004).ADS 
PubMed 
Article 

Google Scholar 
23.Hawkins, E. R. Geographic variations in the whistles of bottlenose dolphins (Tursiops aduncus) along the east and west coasts of Australia. J. Acoust. Soc. Am. 128, 924–935 (2010).ADS 
CAS 
PubMed 
Article 

Google Scholar 
24.Wang, D., Würsig, B. & Evans, W. Whistles of bottlenose dolphins: Comparisons among populations. Aquat. Mamm. 21, 65–77 (1995).
Google Scholar 
25.Connor, R. C., Wells, R. S., Mann, J. & Read, A. J. The bottlenose dolphin: Social relationships in a fission–fusion society. In Cetacean Societies: Field Studies of Dolphins and Whales 91–126 (The University of Chicago Press, 2000).
Google Scholar 
26.Costa, A. P. B. et al. Ecological divergence and speciation in common bottlenose dolphins in the western South Atlantic. J. Evol. Biol. 34, 16–32 (2021).PubMed 
Article 

Google Scholar 
27.Hoelzel, A. R., Potter, C. W. & Best, P. B. Genetic differentiation between parapatric “nearshore” and “offshore” populations of the bottlenose dolphin. Proc. R. Soc. Lond. B 265, 1177–1183 (1998).CAS 
Article 

Google Scholar 
28.Louis, M. et al. Habitat-driven population structure of bottlenose dolphins, Tursiops truncatus, in the North-East Atlantic. Mol. Ecol. 23, 857–874 (2014).PubMed 
Article 

Google Scholar 
29.Wells, R. S., Natoli, A. & Braulik, G. Tursiops truncatus. The IUCN Red List of Threatened Species (2019).30.Marino, L. et al. Cetaceans have complex brains for complex cognition. PLoS Biol. 5, 966–972 (2007).CAS 
Article 

Google Scholar 
31.Janik, V. & Slater, P. Context-specific use suggests that bottlenose dolphin signature whistles are cohesion calls. Anim. Behav. 56, 829–838 (1998).CAS 
PubMed 
Article 

Google Scholar 
32.Sayigh, L. et al. Individual recognition in wild bottlenose dolphins: a field test using playback experiments. Anim. Behav. 57, 41–50 (1999).CAS 
PubMed 
Article 

Google Scholar 
33.Au, W. W. L. Echolocation signals of wild dolphins. Acoust. Phys. 50, 454–462 (2004).ADS 
Article 

Google Scholar 
34.Herzing, D. & dos Santos, M. E. Functional aspects of echolocation in dolphins. In Echolocation in Bats and Dolphins 386–393 (The University of Chicago Press, 2004).
Google Scholar 
35.Jensen, F. H., Bejder, L., Wahlberg, M. & Madsen, P. T. Biosonar adjustments to target range of echolocating bottlenose dolphins (Tursiops sp.) in the wild. J. Exp. Biol. 212, 1078–1086 (2009).CAS 
PubMed 
Article 

Google Scholar 
36.Diáz-López, B. & Shirai, J. Mediterranean common bottlenose dolphin’s repertoire and communication use. In Dolphins: Anatomy, Behavior, and Threats 129–148 (Nova Science Publishers, 2009).
Google Scholar 
37.Herzing, D. L. Acoustics and social behavior of wild dolphins: Implications for a sound society. In Hearing by Whales and Dolphins Springer Handbook of Auditory Research 225–272 (Springer, 2000).
Google Scholar 
38.dos Santos, M. E., Ferreira, A. J. & Harzen, S. Rhythmic sound sequences emitted by aroused bottlenose dolphins in the Sado estuary, Portugal. In Sensory Systems of Aquatic Mammals 325–334 (De Spil Publishers, 1995).
Google Scholar 
39.Luís, A. R., Alves, I. S., Sobreira, F. V., Couchinho, M. N. & dos Santos, M. E. Brays and bits: Information theory applied to acoustic communication sequences of bottlenose dolphins. Bioacoustics 28, 286–296 (2019).Article 

Google Scholar 
40.Jones, B., Zapetis, M., Samuelson, M. M. & Ridgway, S. Sounds produced by bottlenose dolphins (Tursiops): A review of the defining characteristics and acoustic criteria of the dolphin vocal repertoire. Bioacoustics 29(4), 399–440 (2020).Article 

Google Scholar 
41.May-Collado, L. J. & Wartzok, D. A. comparison of bottlenose dolphin whistles in the Atlantic ocean: Factors promoting whistle variation. J. Mammal. 89, 1229–1240 (2008).Article 

Google Scholar 
42.Jones, G. J. & Sayigh, L. S. Geographic variation in rates of vocal production of free-ranging bottlenose dolphins. Mar. Mamm. Sci. 18, 374–393 (2002).Article 

Google Scholar 
43.La Manna, G. et al. Assessing geographical variation on whistle acoustic structure of three Mediterranean populations of common bottlenose dolphin (Tursiops truncatus). Behaviour 154, 583–607 (2017).Article 

Google Scholar 
44.Papale, E. et al. Acoustic divergence between bottlenose dolphin whistles from the Central-Eastern North Atlantic and Mediterranean Sea. Acta Ethologica 17, 155–165 (2014).Article 

Google Scholar 
45.R Development Core Team. R: A Language and Environment for Statistical Computing (2018).46.Wickham, H. ggplot2: Elegant Graphics for Data Analysis. https://ggplot2.tidyverse.org (Springer, 2016).47.Mccomb, K. & Semple, S. Coevolution of vocal communication and sociality in primates. Biol. Lett. 1, 381–385 (2005).PubMed 
PubMed Central 
Article 

Google Scholar 
48.Leighton, G. M. Cooperative breeding influences the number and type of vocalizations in avian lineages. Proc. R. Soc. B Biol. Sci. 284, 1–9 (2017).
Google Scholar 
49.Freeberg, T. M., Dunbar, R. I. M. & Ord, T. J. Social complexity as a proximate and ultimate factor in communicative complexity. Philos. Trans. R. Soc. B Biol. Sci. 367, 1785–1801 (2012).Article 

Google Scholar 
50.Pollard, K. A. & Blumstein, D. T. Evolving communicative complexity: insights from rodents and beyond. Philos. Trans. R. Soc. B Biol. Sci. 367, 1869–1878 (2012).Article 

Google Scholar 
51.Gustison, M. L., Le Roux, A. & Bergman, T. J. Derived vocalizations of geladas (Theropithecus gelada) and the evolution of vocal complexity in primates. Philos. Trans. R. Soc. B Biol. Sci. 367, 1847–1859 (2012).Article 

Google Scholar 
52.Augusto, J. F., Rachinas-Lopes, P. & dos Santos, M. E. Social structure of the declining resident community of common bottlenose dolphins in the Sado Estuary, Portugal. J. Mar. Biol. Assoc. U. K. 92, 1773–1782 (2012).Article 

Google Scholar 
53.Luís, A. R., Couchinho, M. N. & dos Santos, M. E. Changes in the acoustic behavior of resident bottlenose dolphins near operating vessels. Mar. Mamm. Sci. 30, 1417–1426 (2014).Article 

Google Scholar 
54.Ridgway, S. H., Moore, P. W., Carder, D. A. & Romano, T. A. Forward shift of feeding buzz components of dolphins and belugas during associative learning reveals a likely connection to reward expectation, pleasure and brain dopamine activation. J. Exp. Biol. 217, 2910–2919 (2014).CAS 
PubMed 
Article 

Google Scholar 
55.Luís, A. R., Couchinho, M. N. & dos Santos, M. E. A quantitative analysis of pulsed signals emitted by wild bottlenose dolphins. PLoS ONE 11, 1–11 (2016).
Google Scholar 
56.Nowacek, D. P. Acoustic ecology of foraging bottlenose dolphins (Tursiops truncatus) habitat-specific use of three sound types. Mar. Mamm. Sci. 21, 587–602 (2005).Article 

Google Scholar 
57.Caldwell, M. C., Caldwell, D. K. & Tyack, P. L. Review of the signature-whistle-hypothesis for the Atlantic bottlenose dolphin, Tursiops truncatus. In The Bottlenose Dolphin 199–234 (Academic Press, 1990).
Google Scholar 
58.Laland, K. N. & Janik, V. M. The animal cultures debate. Evolution 21, 542–547 (2006).
Google Scholar 
59.Kershenbaum, A., Sayigh, L. S. & Janik, V. M. The encoding of individual identity in dolphin signature whistles: How much information is needed?. PLoS ONE 8, e77671 (2013).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
60.King, S. L. & Janik, V. M. Bottlenose dolphins can use learned vocal labels to address each other. Proc. Natl. Acad. Sci. U.S.A. 110, 13216–13221 (2013).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
61.Sayigh, L., Esch, H., Wells, R. & Janik, V. Facts about signature whistles of bottlenose dolphins, Tursiops truncatus. Anim. Behav. 74, 1631–1642 (2007).Article 

Google Scholar 
62.Buckstaff, K. C. Effects of watercraft noise on the acoustic behavior of bottlenose dolphins, Tursiops truncatus, in Sarasota Bay, Florida. Mar. Mamm. Sci. 20, 709–725 (2004).Article 

Google Scholar 
63.Morisaka, T., Shinohara, M., Nakahara, F. & Akamatsu, T. Geographic variations in the whistles among three Indo-Pacific bottlenose dolphin. Fish. Sci. 71, 568–576 (2005).CAS 
Article 

Google Scholar 
64.May-Collado, L. J. & Quiñones-Lebrón, S. G. Dolphin changes in whistle structure with watercraft activity depends on their behavioral state. J. Soc. Am. 135, EL193–EL198 (2014).ADS 

Google Scholar 
65.Garland, E. C. et al. Report dynamic horizontal cultural transmission of humpback whale song at the ocean basin scale. Curr. Biol. 21, 687–691 (2011).CAS 
PubMed 
Article 

Google Scholar 
66.Whitehead, H. & Rendell, L. The Cultural Lives of Whales and Dolphins (The University of Chicago Press, 2015).
Google Scholar 
67.Herzing, D. L. Vocalizations and associated underwater behavior of free-ranging Atlantic spotted dolphins, Stenella frontalis and bottlenose dolphins, Tursiops truncatus. Aquat. Mamm. 22, 61–79 (1996).
Google Scholar 
68.May-Collado, L. J. Changes in whistle structure of two dolphin species during interspecific associations. Ethology 116, 1065–742010 (2010).Article 

Google Scholar 
69.Catchpole, C. K. The evolution of bird sounds in relation to mating and spacing behavior. In Acoustic Communication in Birds 297–319 (Academic Press, 1982).
Google Scholar 
70.Herman, L. M. The multiple functions of male song within the humpback whale (Megaptera novaeangliae) mating system: Review, evaluation, and synthesis. Biol. Rev. 92, 1795–1818 (2017).PubMed 
Article 

Google Scholar 
71.Janik, V. M. Food-related bray calls in wild bottlenose dolphins (Tursiops truncatus). Proc. R. Soc. B Biol. Sci. 267, 923–927 (2000).CAS 
Article 

Google Scholar 
72.King, S. L. & Janik, V. M. Come dine with me: food-associated social signalling in wild bottlenose dolphins (Tursiops truncatus). Anim. Cogn. 18, 969–974 (2015).PubMed 
Article 

Google Scholar 
73.Herzing, D. L. Synchronous and rhythmic vocalizations and correlated underwater behavior of free-ranging Atlantic Spotted Dolphins (Stenella frontalis) and Bottlenose Dolphins (Tursiops truncatus) in the Bahamas. Anim. Behav. Cogn. 2, 14–29 (2015).Article 

Google Scholar 
74.Pleslić, G. et al. The abundance of common bottlenose dolphins (Tursiops truncatus) in the former special marine reserve of the Cres-Lošinj Archipelago, Croatia. Aquat. Conserv. Mar. Freshwat. Ecosyst. 25, 125–137 (2015).Article 

Google Scholar 
75.Rako-Gospic, N., Radulovi, M., Vu, T., Plesli, G. & Mackelworth, P. Factor associated variations in the home range of a resident Adriatic common bottlenose dolphin population. Mar. Pollut. Bull. 124, 234–244 (2017).CAS 
PubMed 
Article 

Google Scholar 
76.Rako, N. et al. Leisure boating noise as a trigger for the displacement of the bottlenose dolphins of the Cres-Lošinj archipelago (northern Adriatic Sea, Croatia). Mar. Pollut. Bull. 68, 77–84 (2013).CAS 
PubMed 
Article 

Google Scholar 
77.Barragán-Barrera, D. C. et al. High genetic structure and low mitochondrial diversity in bottlenose dolphins of the Archipelago of Bocas del Toro, Panama: A population at risk?. PLoS ONE 12, 1–22 (2017).Article 
CAS 

Google Scholar 
78.Ey, E. & Fischer, J. The, “Acoustic Adaptation Hypothesis”—A review of the evidence from birds, anurans and mammals. Bioacoustics 19, 21–48 (2009).Article 

Google Scholar 
79.Papale, E., Azzolin, M. & Giacoma, C. Vessel traffic affects bottlenose dolphin (Tursiops truncatus) behaviour in waters surrounding Lampedusa Island, south Italy. J. Mar. Biol. Assoc. U.K. 92, 1877–1885 (2012).Article 

Google Scholar 
80.Gridley, T., Nastasi, A., Kriesell, H. J. & Elwen, S. H. The acoustic repertoire of wild common bottlenose dolphins (Tursiops truncatus) in Walvis Bay, Namibia. Bioacoustics 24, 153–174 (2015).Article 

Google Scholar 
81.Au, W. W. L. & Hastings, M. C. Emission of social sounds by marine animals. In Principles of Marine Bioacoustics 401–499 (Springer, 2008).
Google Scholar 
82.Bázua-Duran, C. & Bazúa-Durán, C. Differences in the whistle characteristics and repertoire of Bottlenose and Spinner Dolphins. An. Acad. Bras. Ciênc. 76, 386–392 (2004).PubMed 
Article 

Google Scholar 
83.Lammers, M. O., Au, W. W. L. & Herzing, D. L. The broadband social acoustic signaling behavior of spinner and spotted dolphins. J. Acoust. Soc. Am. 114, 1629–1639 (2003).ADS 
PubMed 
Article 

Google Scholar 
84.Simard, P. et al. Low frequency narrow-band calls in bottlenose dolphins (Tursiops truncatus): Signal properties, function, and conservation implications. J. Acoust. Soc. Am. 130, 3068–3076 (2011).ADS 
PubMed 
Article 

Google Scholar 
85.Luís, A. R., Couchinho, M. N. & dos Santos, M. E. Signature whistles in wild bottlenose dolphins: Long-term stability and emission rates. Acta Ethologica 19, 113–122 (2016).Article 

Google Scholar 
86.Ford, J. K. B. Vocal traditions among resident killer whales (Orcinus orca) in coastal waters of British Columbia. Can. J. Zool. 69, 1454–1483 (1991).Article 

Google Scholar 
87.Papale, E. et al. Biphonic calls as signature whistles in a free-ranging bottlenose dolphin. Bioacoustics 24, 223–231 (2015).Article 

Google Scholar 
88.Elliser, C. R. & Herzing, D. L. Long-term interspecies association patterns of Atlantic bottlenose dolphins, Tursiops truncatus, and Atlantic spotted dolphins, Stenella frontalis, in the Bahamas. Mar. Mamm. Sci. 32, 38–56 (2015).Article 

Google Scholar 
89.Hoffmann-Kuhnt, M., Herzing, D. L., Ho, A. & Chitre, M. A. Whose line sound is it anyway? Identifying the vocalizer on underwater video by localizing with a hydrophone array. Anim. Behav. Cogn. 3, 288–298 (2016).Article 

Google Scholar 
90.Lima, I. M. S. et al. Whistle comparison of four delphinid species in Southeastern Brazil. J. Acoust. Soc. Am. 139, EL124 (2016).ADS 
PubMed 
Article 

Google Scholar  More

We performed shotgun metagenome (assessing functional diversity) and 16S rRNA gene V4 amplicon (assessing taxonomic diversity) sequencing of time-series samples from the closed laboratory mesocosm chambers with oil addition (oiled) or without (control) to test whether or not the specialization disturbance hypothesis could explain the microbial community succession patterns (response). Additionally, metagenomic datasets from the Pensacola Beach field study [4] were included for comparison. The latter datasets represented beach sands before the oil had reached the coast (Pre-Spill), while the beach was contaminated (Spill-Oiled and Weathered), and after the oil concentrations in beach sands had reached undetectable levels (Recovered) (Fig. 1). The detailed description of the sample processing, sequencing, and bioinformatic analyses can be found in the Supplementary Online material (Figs. S3 and S4). Nonpareil, a tool that estimates what fraction of the microbial community is represented in a metagenome (i.e., the coverage) by examining the level of redundancy among the metagenomic reads [13], showed that coverage of the sampled microbial communities by sequencing was adequate for comparison [14], with 60–75% sample coverage for oiled mesocosm and 45–70% for control sample. In addition, Nonpareil sequence diversity (Nd), an estimate of the total diversity in sequence space harbored by a microbial community, and other diversity metrics showed that control samples (no oil added) harbored higher diversity. Applying the commonly used pipeline of assigning 16S rRNA gene fragments recovered in the metagenomes against the SILVA database release 132 (ref. [15]) using VSEARCH in QIIME2 [16] and 97% nucleotide identity for a match (closed OTU picking) resulted in 11% fewer reads assigned for control vs. oiled mesocosm metagenomes and 27% fewer reads assigned for clean vs. oiled Pensacola metagenomes.These results indicated that the control samples potentially harbored more novel (uncharacterized) taxa that could confound taxonomic comparisons due to the comparatively lower number of taxonomically identified sequences. To account for this effect, we employed a manual pipeline with BLASTn [17], and a lower cut-off (90% nucleotide identity) for read assignment to the database (Method 2). Additionally, we performed our analysis based on both 16S rRNA gene amplicon sequences as well as 16S-carrying metagenomic reads, and employed Hill numbers, represented as qD, a group of diversity indices that take into account species abundance and richness to compute the equivalent number of species at an order q, where q adjusts the sensitivity to rare species (see also [18] and references therein). Our results, after rarefying the 16S rRNA gene fragment OTU abundance to the metagenomic dataset with the lowest coverage [18], showed that the inverse Simpson index (2D) was lower in oiled chambers with a mean of 274 (SD = 146) than in control chambers with a mean of 896 (SD = 86; Table 1). The Welch’s t-test revealed a significant difference at alpha 0.05 (p value = 8.89e−4). Amplicon data from the same mesocosm samples (Fig. 1) or analysis at the sequence variant level (ASVs; Fig. S5) showed similar results (Fig. 1). See supplementary results and discussion for further details (Fig S6).Table 1 Summary statistics and hypothesis testing for functional diversity and species diversity indices.Full size tableFunctional diversity was analyzed based on the number of metagenomic reads matching molecular function gene ontology (GO) terms [19] as previously described [7]. Our analysis showed that functional diversity (1D) was higher in oiled chambers with a mean of 193 equivalent GO terms (SD = 18) compared to control chambers with a mean of 105 equivalent GO terms (SD = 31; Table 1; Chao-Shen Entropy Estimator; p value = 1.14e-05, two-tailed Welch’s t-test).Collectively, the results presented here from closed system mesocosms, which were designed to limit fluctuations in environmental conditions, stochasticity, and dispersal, showed that the specialization disturbance hypothesis can explain microbial succession patterns following crude oil perturbations in coastal beach sand environments. Recent incubation experiments of microbial communities from sandy soils have also provided evidence in support of the specialization-disturbance hypothesis (preprint available at the time of writing [6]), and the close agreement of these results with those of previous field observations (e.g., Table 1, Fig. 1) [7] suggested that this underlying explanation/mechanism is robust even in light of environmental variation and drift. The high similarity in taxonomic composition between our mesocosms and our previous field data also suggested that the key oil degraders were present in the clean sands at the time of sampling for establishing the mesocosms, 7 years after the DWH oil spill. The survival strategy of oil degraders in the clean sand remains an interesting question, and has implications for whether or not their niche breadth includes uncontaminated sandy sediments. It would be interesting to test whether similar patterns are observed in other habitats (e.g., beach sand from an alternative source lacking a history of oil exposure) and other types of perturbation in order to test the universal applicability of the results reported here. With sufficient background data available on the unperturbed ecosystem, we believe that the approach outlined here based on specialist vs. generalist taxa should be able to elucidate whether or not the specialization disturbance can explain microbial responses to other types of perturbations and/or identify recovered ecosystems. More

1.

2.Amoabeng, B. W. et al. Tri-trophic insecticidal effects of African plants against cabbage pests. PloS. One. 8(10), (2013).3.Hasan, F. & Ansari, M. S. Effect of different cole crops on the biological parameters of Pieris brassicae (L.)(Lepidoptera: Pieridae) under laboratory conditions. J. Crop Sci. Biotechnol. 13(3), 195–202 (2010).Article 

Google Scholar 
4.Yang, H., Piao, X., Zhang, L., Song, S. & Xu, Y. Ginsenosides from the stems and leaves of Panax ginseng show antifeedant activity against Plutella xylostella (Linnaeus). Ind. Crops. Prod. 124, 412–417 (2018).CAS 
Article 

Google Scholar 
5.Mazhawidza, E. & Mvumi, B. M. Field evaluation of aqueous indigenous plant extracts against the diamondback moth, Plutella xylostella L. and the rape aphid, Brevicoryne brassicae L. in brassica production. Ind. Crop. Prod. 110, 36–44 (2017).Article 

Google Scholar 
6.Akhtar, Y., Isman, M. B., Niehaus, L. A., Lee, C. H. & Lee, H. S. Antifeedant and toxic effects of naturally occurring and synthetic quinones to the cabbage looper. Trichoplusia ni. Crop Prot. 31(1), 8–14 (2012).CAS 
Article 

Google Scholar 
7.Aktar, W., Sengupta, D. & Chowdhury, A. Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2(1), 1–12 (2009).PubMed 
PubMed Central 
Article 

Google Scholar 
8.Couto, I. F. S. et al. Botanical extracts of the brazilian savannah affect feeding and oviposition of Plutella xylostella (Linnaeus, 1758)(Lepidoptera: Plutellidae). J. Agric. Sci. 11(5), (2019).9.Lengai, G. M., Muthomi, J. W. & Mbega, E. R. Phytochemical activity and role of botanical pesticides in pest management for sustainable agricultural crop production. Sci. Afr. 7, e00239 (2020).
Google Scholar 
10.Hikal, W. M., Baeshen, R. S. & Said-Al Ahl, H. A. Botanical insecticide as simple extractives for pest control. Cogent. Biol. 3(1), 1404274 (2017).Article 
CAS 

Google Scholar 
11.Isman, M. B. Botanical insecticides, deterrents, and repellent in modern agriculture and an increasingly regulated world. Ann. Rev. Entomol. 51, 45–66 (2006).CAS 
Article 

Google Scholar 
12.Petacci, F. et al. Phytochemistry and quantification of polyphenols in extracts of the Asteraceae weeds from Diamantina, Minas Gerais State Brazil. Planta. Daninha. 30, 9–15 (2012).Article 

Google Scholar 
13.Furlong, M. J., Wright, D. J. & Dosdall, L. M. Diamondback moth ecology and management: problems, progress, and prospects. Annu. Rev. Entomol. 58, 517–541 (2013).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
14.Sulifoa, J. B., Fangupo, S. & Kant, R. Oviposition periodicity, egg morphology and life history of large cabbage moth Crocidolomia pavonana population in Samoa. SPJNAS 34(2), 29–34 (2016).
Google Scholar 
15.Lal, M. N., & Bhajan Ram. Cabbage butterfly, Pieris brassicae L.-an upcoming menace for Brassica oilseed crops in Northern India. Cruciferae Newsletter 25. (2004).16.Chalfant, R. B., Denton, W. H., Schuster, D. J. & Workman, R. B. Management of cabbage caterpillars in Florida and Georgia by using visual damage thresholds. J. Econ. Entomol. 72, 411–413 (1979).Article 

Google Scholar 
17.Lim, G. S., Sivapragasam, A., & Loke, W. H. Crucifer insect pest problems: trends, issues and management strategies. In The Management of diamondback and other crucifer pests. Proceedings of the third international workshop, Kuala Lumpur, Malaysia. (1996).18.Tumutegyereize, J. K. Handbook on identification and management of pests and diseases of cabbage and other brassicas in Uganda. (2008).19.Amoabeng, B. W., Johnson, A. C. & Gurr, G. M. Natural enemy enhancement and botanical insecticide source: a review of dual use companion plants. Appl. Entomol. Zool. 54(1), 1–19 (2019).Article 

Google Scholar 
20.Gurr, G. M., Steve, D. W., Douglas, A. L. & Minsheng, Y. Habitat management to suppress pest populations: progress and prospects. Annu. Rev. Entomol. 62, 91–109 (2017).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
21.Warwick, H. R. I., & Wellesbourne, W. Control of Diamond back Moth (Plutella xylostella) on Cabbage (Brassica oleracea var capitata) using Intercropping with Non-Host Crops “E. Asare-Bediako,” AA Addo-Quaye and A. Mohammed” Department of Crop Science, University of Cape Coast, Cape Coast, Ghana. Am. J. Food Technol. 5(4), 269–274 (2010).Article 

Google Scholar 
22.Xu, Q. C. et al. Relay-intercropping into tomato decreases cabbage pest incidence. J Food. Agric. Environ. 8, 1037–1041 (2010).
Google Scholar 
23.Yarou, B. B et al. Efficacy of Basil-Cabbage intercropping to control insect pests in Benin, West Africa. Commun. Agric. Biol. 82, 157–166(2017)24.Shukla, A. & Kumar, A. The diamond back moth, Plutella xylostella a problematic pest of Brassica crop. J. Adv. Indian. Entomol. 1, 229–240 (2005).
Google Scholar 
25.Olesen, J. E. & Bindi, M. Consequences of climate change for European agricultural productivity, land use and policy. Eur. J. Agron. 16, 239–262. https://doi.org/10.1016/S1161-0301(02)00004-7 (2002).Article 

Google Scholar 
26.Dey, D., Routray, S., Baral, S. & Mahantheshwara, B. Effect of planting dates and botanical insecticides against major Lepidopterous pests of cabbage: a review. Agric. Rev. 38(1), 60–66 (2017).
Google Scholar 
27.Tanyi, C. B., Ngosong, C. & Ntonifor, N. N. Effects of climate variability on insect pests of cabbage: adapting alternative planting dates and cropping pattern as control measures. Chem. Biol. Technol. Agric. 5(1), 25 (2018).Article 

Google Scholar 
28.Viraktamath, S., Shekarappa Reddy, B. S. & Patil, M. G. Effect of date of planting on the extent of damage by the Diamond back moth, Plutella xylostella on cabbage. Karnataka. J. Agric. Sci. 7, 238–239 (1994).
Google Scholar 
29.Pickett, J. A., Christine, M. W., Charles, A. O. M. & Zeyaur, R. K. Push–pull farming systems. Curr. Opi. Biotech. 26, 125–132 (2014).CAS 
Article 

Google Scholar 
30.Kergunteuil, A., Dugravot, S., Danner, H., Van Dam, N. M. & Cortesero, A. M. Characterizing volatiles and attractiveness of five brassicaceous plants with potential for a ‘push-pull’strategy toward the cabbage root fly Delia radicum. J. Chem. Ecol. 41(4), 330–339 (2015).CAS 
PubMed 
Article 

Google Scholar 
31.Khan, Z. R. et al. Achieving food security for one million subSaharan African poor through push—pull innovation by 2020. Philos. Trans. R. Soc. B 369, 20120284 (2014).Article 

Google Scholar 
32.Cook, S. M., Khan, Z. R. &Pickett, J. A. The use of push-pull strategies in integrated pest management. Annu. Rev. Entomol. 52, 375–400 (2007).CAS 
PubMed 
Article 

Google Scholar 
33.Khan, Z. R. et al. Intercropping increases parasitism of pests. Nature 388, 631–632 (1997).ADS 
CAS 
Article 

Google Scholar 
34.Khan, Z. R., Pickett, J. A., Berg, J. V. D., Wadhams, L. J. & Woodcock, C. M. Exploiting chemical ecology and species diversity: stem borer and striga control for maize and sorghum in Africa. Pest. Manag. Sci. 56, 957–962 (2000).CAS 
Article 

Google Scholar 
35.Parolin, P. et al. Secondary plants used in biological control: a review. Int. J. Pest Manag. 58(2), 91–100 (2012).Article 

Google Scholar 
36.Teal P.E.A. Sex attractant pheromones. In: Encyclopedia of Entomology. https://doi.org/10.1007/0-306-48380-7_3866 (Springer, Dordrecht, 2004)37.Witzgall, P., Lindblom, T., Bengtsson, M., & Toth, M. The Pherolist. (2004) http://www.pherolist.slu.se/pherolist.php. Accessed 23 July 201338.Witzgall, P., Stelinski, L., Gut, L. & Thomson, D. Codling moth management and chemical ecology. Ann Rev Entomol. 53(1), 503–522 (2008).CAS 
Article 

Google Scholar 
39.Schroeder, P. C., Shelton, A. M., Ferguson, C. S., Hoffmann, M. P. & Petzoldt, C. H. Application of synthetic sex pheromone for management of diamondback moth, Plutella xylostella, in cabbage. Entomol Exp. Appl. 94(3), 243–248 (2000).CAS 
Article 

Google Scholar 
40.Reddy, G. V. & Guerrero, A. New pheromones and insect control strategies. In Vitamins & hormones 83, 493–519. (Academic Press, 2010).41.Reddy, G. P. & Urs, K. D. Mass trapping of diamondback moth Plutella xylostella in cabbage fields using synthetic sex pheromones. Int. J. Pest Manag. 39(4), 125–126 (1997).
Google Scholar 
42.Isman, M. B. & Botanical insecticides, ,. deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu. Rev. Entomol. 51, 45 (2006).CAS 
PubMed 
Article 

Google Scholar 
43.Isman, M. B. & Grieneisen, M. L. Botanical insecticide research: many publications, limited useful data. Trends Plant. Sci. 19(3), 140–145 (2014).CAS 
PubMed 
Article 

Google Scholar 
44.Campos, E. V. et al. Use of botanical insecticides for sustainable agriculture: Future perspectives. Ecol. Indic. 105, 483–495 (2019).CAS 
Article 

Google Scholar 
45.El-Wakeil, N. E. Retracted Article: Botanical Pesticides and Their Mode of Action. Gesunde Pflanzen 65(4), 125–149 (2013).CAS 
Article 

Google Scholar 
46.Lengai, G. M., Muthomi, J. W. & Mbega, E. R. Phytochemical activity and role of botanical pesticides in pest management for sustainable agricultural crop production. Sci. Afr. 7, e00239 (2020).
Google Scholar 
47.Bennett, R. N. & Wallsgrove, R. M. Secondary metabolites in plant defence mechanisms. New Phytol. 127, 617–633 (1994).CAS 
PubMed 
Article 

Google Scholar 
48.Samarasekera, J. Insecticidal natural products from Sri Lankan plants (Doctoral dissertation, The Open University) (1997).49.Bambawale, O. M., & Bhagat, S. O. M. E. S. H. W. A. R. Registration related issues in effective use of biopesticides in pest management. Biopesticides in environment and food security: Issuers and strategies, 265–285 (2012).50.Isman, M. B. A renaissance for botanical insecticides?. Pest Manag. Sci. 71(12), 1587–1590 (2015).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
51.Begum, S., Wahab, A., Siddiqui, B. S. & Qamar, F. Nematicidal Constituents of the aerial parts of Lantana camara. J. Nat. Prod. 63, 765–767 (2000).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
52.Baidoo, P. K. & Adam, J. I. The effects of extracts of Lantana camara (L.) and Azadirachta indica (A. Juss) on the population dynamics of Plutella xylostella, Brevicoryne brassicae and Hellula undalis on cabbage (2012).53.Kumar, K. K. et al. Microbial biopesticides for insect pest management in India: current status and future prospects. J. Invertebr. Pathol. 165, 74–81 (2019).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
54.Reddy, S. E., Dolma, S. K. & Bhardwaj, A. Plants of himalayan region as potential source of biopesticides for lepidopteran insect pests. Springer, New Delhi. In Herbal Insecticides, Repellents and Biomedicines: Effectiveness and Commercialization, 63–83 (2016).55.Dougoud, J., Toepfer, S., Bateman, M. & Jenner, W. H. Efficacy of homemade botanical insecticides based on traditional knowledge A review. Agron. Sustain. Dev. 39(4), 37 (2019).Article 
CAS 

Google Scholar 
56.Sarasan, V., Kite, G. C., Sileshi, G. W. & Stevenson, P. C. Applications of phytochemical and in vitro techniques for reducing over-harvesting of medicinal and pesticidal plants and generating income for the rural poor. Plant Cell Rep. 30, 1163 (2011).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
57.Figueiredo, A.C., Barroso, J.G., Pedro. L.G., Scheffer, J.J.C. Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour Fragr J. 23(4), 213 (2008)58.Yakkundi, S. R., Thejavathi, R. & Ravindranath, B. Variation of azadirachtin content during growth and storage of neem (Azadirachta indica) seeds. J. Agric. Food Chem. 43(9), 2517 (1995).CAS 
Article 

Google Scholar 
59.Tak, J. H. & Isman, M. B. Penetration-enhancement underlies synergy of plant essential oil terpenoids as insecticides in the cabbage looper Trichoplusia ni. Sci. Rep. 7, 42432 (2017).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
60.Tak, J. H. & Isman, M. B. Enhanced cuticular penetration as the mechanism for synergy of insecticidal constituents of rosemary essential oil in Trichoplusia ni. Sci. Rep. 5(1), 1–10 (2015).
Google Scholar 
61.Tak, J. H., Jovel, E. & Isman, M. B. Contact, fumigant, and cytotoxic activities of thyme and lemongrass essential oils against larvae and an ovarian cell line of the cabbage looper Trichoplusia ni. J. Pest. Sci. 89(1), 183–193 (2016).Article 

Google Scholar 
62.Pavela, R. Acute, synergistic and antagonistic effects of some aromatic compounds on the Spodoptera littoralis Boisd. (Lep, Noctuidae) larvae. Ind Crops Prod 60, 247–258 (2014)63.Arthurs, S. & Dara, S. K. Microbial biopesticides for invertebrate pests and their markets in the United States. J. Invertebr. Pathol. 165, 13–21 (2019).PubMed 
Article 
PubMed Central 

Google Scholar 
64.NBAIR. ICAR-National Bureau of Agricultural Insect Resources, Newsletter,. Bengaluru. India 9(4p), 2017 (2017).
Google Scholar 
65.Maina, U. M., Galadima, I. B., Gambo, F. M. & Zakaria, D. A review on the use of entomopathogenic fungi in the management of insect pests of field crops. J. Entomol. Zool. Stud. 6(1), 27–32 (2018).
Google Scholar 
66.Sujeetha, J. A. R., & Sahayaraj, K. Role of entomopathogenic fungus in pest management. In Basic and applied aspects of biopesticides (pp. 31–46). Springer, New Delhi. (2014).67.Glare, T. R., Jurat-Fuentes, J. L. & O’callaghan, M. Basic and applied research: entomopathogenic bacteria. In Microbial control of insect and mite pests 47–67. (Academic Press, 2017).68.Van Frankenhuyzen, K. Insecticidal activity of Bacillus thuringiensis crystal proteins. J. Invertebr. Pathol. 101(1), 1–16 (2009).PubMed 
Article 
CAS 
PubMed Central 

Google Scholar 
69.Arora, N. K., Khare, E. & Maheshwari, D. K. Plant growth promoting rhizobacteria: constraints in bioformulation, commercialization, and future strategies. In Plant growth and health promoting bacteria 97–116 (Springer, Berlin, Heidelberg 2010).70.Gupta, S. & Dikshit, A. K. Biopesticides: An ecofriendly approach for pest control. J. Biopestic., 3(Special Issue), 186 (2010).71.Lacey, L. A., Frutos, R., Kaya, H. K. & Vail, P. Insect pathogens as biological control agents: do they have a future?. Biol. Control. 21(3), 230–248 (2001).Article 

Google Scholar 
72.Mishra, J., Tewari, S., Singh, S. & Arora, N. K. Biopesticides: where we stand? In Plant microbes symbiosis: Applied Facets, 37-75. (Springer. New Delhi, 2015)73.Aneja, K. R., Khan, S. A. & Aneja, A. Biopesticides an eco-friendly pestmanagement approach in agriculture: status and prospects. Kavaka 47, 145–154 (2016).
Google Scholar 
74.Kumar, R. et al. Chemical composition, cytotoxicity and insecticidal activities of Acorus calamus accessions from the western Himalayas. Ind. Crops. Prod. 94, 520–527 (2016).CAS 
Article 

Google Scholar 
75.Rioba, N. B. & Stevenson, P. C. Ageratum conyzoides L for the management of pests and diseases by small holder farmers. Ind. Crops. Prod. 110, 22–29 (2017).Article 

Google Scholar 
76.Datta, R., Kaur, A., Saraf, I., Singh, I. P. & Kaur, S. Effect of crude extracts and purified compounds of Alpinia galanga on nutritional physiology of a polyphagous lepidopteran pest, Spodoptera litura (Fabricius). Ecotoxicol. Environ. 168, 324–329 (2019).CAS 
Article 

Google Scholar 
77.Hwang, K. S., Kim, Y. K., Kim, Y. T., Lee, J. & Park, K. W. A tetracosatetraene as larvicidal compound isolated from Alpinia katsumadai. Ind. Crops. Prod. 109, 786–789 (2017).CAS 
Article 

Google Scholar 
78.Castillo-Sánchez, L. E., Jiménez-Osornio, J. J. & Delgado-Herrera, M. A. Secondary metabolites of the Annonaceae, Solanaceae and Meliaceae families used as biological control of insects. Trop. Subtrop. Agroecosyst. 12(3), 445–462 (2010).
Google Scholar 
79.Leatemia, J. A. & Isman, M. B. Efficacy of crude seed extracts of Annona squamosa against diamondback moth, Plutella xylostella L. in the greenhouse. Int. J. Pest. Manage. 50(2), 129–133 (2004).Article 

Google Scholar 
80.Okwute, S. K. Plants as potential sources of pesticidal agents: a review. Pesticides-Advances in Chemical and Botanical Pesticides. InTech (2012).81.Torres, A. L., Barros, R. & Oliveira, J. V. D. Effects of plant aqueous extracts on the development of Plutella xylostella (L.)(Lepidoptera: Plutellidae). Neotrop. Entomol. 30(1), 151–156 (2001).Article 

Google Scholar 
82.Sharma, A. & Gupta, R. Biological activity of some plant extracts against Pieris brassicae (Linn.). J. Biopestic. 2(1), 26–31 (2009).CAS 

Google Scholar 
83.Khanavi, M., Laghaei, P. & Isman, M. B. Essential oil composition of three native Persian plants and their inhibitory effects in the cabbage looper Trichoplusia ni. J. Asia-Pac. Entomol. 20(4), 1234–1240 (2017).Article 

Google Scholar 
84.Ma, S., Jia, R., Guo, M., Qin, K. & Zhang, L. Insecticidal activity of essential oil from Cephalotaxus sinensis and its main components against various agricultural pests. Ind. Crop. Prod. 150, 112403 (2020).CAS 
Article 

Google Scholar 
85.Yankanchi, S. R. & Patil, S. R. Field efficacy of plant extracts on larval populations of Plutella xylostella L. and Helicoverpa armigera Hub and their impact on cabbage infestation. J. Biopestic. 2(1), 32–36 (2009).CAS 

Google Scholar 
86.Filomeno, C. A. et al. Corymbia spp. and Eucalyptus spp. essential oils have insecticidal activity against Plutella xylostella. Ind. Crops. Prod. 109, 374–383 (2017).CAS 
Article 

Google Scholar 
87.de Souza Tavares, W., Akhtar, Y., Gonçalves, G. L. P., Zanuncio, J. C. & Isman, M. B. Turmeric powder and its derivatives from Curcuma longa rhizomes: insecticidal effects on cabbage looper and the role of synergists. Sci. Rep. 6, 34093 (2016).ADS 
PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
88.Tak, J. H., Jovel, E. & Isman, M. B. Comparative and synergistic activity of Rosmarinus officinalis L essential oil constituents against the larvae and an ovarian cell line of the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae). Pest. Manag. Sci. 72(3), 474–480 (2016).CAS 
PubMed 
Article 

Google Scholar 
89.Sanda, K., Koba, K., Poutouli, W., Idrissou, N. & Agbossou, A. B. Pesticidal propertties of Cymbopogon schoenatus against the Diamondback moth Plutella xylostella L (Lepidoptera: Hyponomeutidae). Discov. Innov. 19, 220–225 (2006).
Google Scholar 
90.Qin, X. P., Zhao, H. Y. & Yang, M. L. Antifeeding activities of Dodonaea viscose seed extracts against Plutella xylostella. Chin. J. Entomol. 4, (2008).91.Jahan, F., Abbasipour, H. & Hasanshahi, G. Fumigant toxicity and nymph production deterrence effect of five essential oils on adults of the cabbage aphid, Brevicoryne brassicae L. (Hemiptera: Aphididae). J. Essent. Oil. Bear. Pl. 19(1), 140–147 (2016).CAS 
Article 

Google Scholar 
92.Adebisi, O., Dolma, S. K., Verma, P. K., Singh, B. & Reddy, S. E. Volatile, non-volatile composition and insecticidal activity of Eupatorium adenophorum Spreng against diamondback moth, Plutella xylostella (L.), and aphid Aphis craccivora Koch. Toxin. Rev. 38(2), 143–150 (2019).CAS 
Article 

Google Scholar 
93.Khan, Z. R. et al. Management of witchweed, Striga hermonthica, and stemborers in sorghum, Sorghum bicolor, through intercropping with greenleaf desmodium Desmodium intortum. Int. J. Pest. Manag. 52, 297–302 (2006).Article 

Google Scholar 
94.Afshar, F. H., Maggi, F., Iannarelli, R., Cianfaglione, K. & Isman, M. B. Comparative toxicity of Helosciadium nodiflorum essential oils and combinations of their main constituents against the cabbage looper, Trichoplusia ni (Lepidoptera). Ind. Crops. Prod. 98, 46–52 (2017).Article 
CAS 

Google Scholar 
95.Pseudaletia unipuncta Bullangpoti, V., Wajnberg, E., Audant, P. & Feyereisen, R. Antifeedant activity of Jatropha gossypifolia and Melia azedarach senescent leaf extracts on Spodoptera frugiperda (Lepidoptera: Noctuidae) and their potential use as synergists. Pest. Manag. Sci. 68(9), 1255–1264 (2012)96.Mvumi, C. & Maunga, P. R. Efficacy of lantana (Lantana camara) extract application against aphids (Brevicoryne brassicae) in rape (Brassica napus) over varied periods of time. Afr. J. Biotechnol. 17(8), 249–254 (2018).CAS 
Article 

Google Scholar 
97.Kumar, R., Sharma, K. C. & Kumar, D. Studies on ovicidal effects of some plant extracts against the diamondback moth, Plutella xylostella (L.) infesting cauliflower crop. Biol. Forum. Int. J. (2009). (Vol. 1, No. 1, 47–50).98.Akhtar, Y., Yeoung, Y. R. & Isman, M. B. Comparative bioactivity of selected extracts from Meliaceae and some commercial botanical insecticides against two noctuid caterpillars Trichoplusia ni and. Phytochem. Rev. 7, 77–88 (2008).CAS 
Article 

Google Scholar 
99.Akhtar, Y. & Isman, M. B. Comparative growth inhibitory and antifeedant effects of plant extracts and pure allelochemicals on four phytophagous insect species. J. App. Entomol. 128(1), 32–38 (2004).CAS 
Article 

Google Scholar 
100.Bandeira, G. N. et al. Insecticidal activity of Muntingia calabura extracts against larvae and pupae of diamondback, Plutella xylostella (Lepidoptera, Plutellidae). J. King Saud Univ. Sci. 25(1), 83–89 (2013).Article 

Google Scholar 
101.Nasr, M., Sendi, J. J., Moharramipour, S. & Zibaee, A. Evaluation of Origanum vulgare L. essential oil as a source of toxicant and an inhibitor of physiological parameters in diamondback moth, Plutella xylustella L. (Lepidoptera: Pyralidae). J. Saudi Soc. Agric. Sci. 16(2), 184–190 (2017).
Google Scholar 
102.Shafiei, F., Ahmadi, K. & Asadi, M. Evaluation of systemic effects of four plant extracts compared with two systemic pesticides, acetamiprid and pirimicarb through leaf spraying against Brevicoryne brassicae L. (Hemiptera: Aphididae). J. Vector Ecol. 30, 284–288 (2018).
Google Scholar 
103.Xu, X. R., Jiang, H. Y., Zhang, Y. N. & Feng, P. Z. Bioactivity of Pharbitis purpurea extracts against Plutella xylostella. Pesticides-Shenyang- 45(2), 125 (2006).CAS 

Google Scholar 
104.Kodjo, T. A. et al. Bio-insecticidal effects of plant extracts and oil emulsions of Ricinus communis L. (Malpighiales: Euphorbiaceae) on the diamondback, Plutella xylostella L. (Lepidoptera: Plutellidae) under laboratory and semi-field conditions. J. Appl. Biosci 43, 2899–2914 (2011).
Google Scholar 
105.Khorrami, F., Soleymanzade, A. & Forouzan, M. Toxicity of some medicinal plant extracts to Pieris brassicae and combined effects with Proteus® against Brevicoryne brassicae. J. Phytopathol. Pest Manag. 50–55 (2017).106.Yankanchi, S. R. & Patil, S. R. Field efficacy of plant extracts on larval populations of Plutella xylostella L. and Helicoverpa armigera Hub. and their impact on cabbage infestation. J. Biopestic. 2(1), 32–36 (2009).CAS 

Google Scholar 
107.Ramanujam, B., Rangeshwaran, R., Sivakmar, G., Mohan, M. & Yandigeri, M. S. Management of insect pests by microorganisms. In Proc Indian Nat Sci Acad (Vol. 80) 2, 455–471 (2014).108.Singh, A., Bhardwaj, R. & Singh, I. K. Biocontrol Agents: Potential of Biopesticides for Integrated Pest Management. In Biofertilizers for Sustainable Agriculture and Environment 413–433 (Springer, Cham. 2019).109.Ghosh, S.K., Chaudhary, M. & Kumar P. Myco-Jaal: a novel formulation of Beauveria bassiana for managing diamondback moth (Plutella xylostella) in tropical and sub-tropical crucifer production systems. Proc. of the Sixth International Workshop on Management of the Diamondback Moth and Other Crucifer Insect Pests, AVRDC – The World Vegetable Center, Tainan, Taiwan. pp. 153–158 (2011) 92110.Srinivasan, R., Sevgan, S., Ekesi, S. & Tamò, M. Biopesticide based sustainable pest management for safer production of vegetable legumes and brassicas in Asia and Africa. Pest Manag. Sci. 75(9), 2446–2454 (2019).CAS 
PubMed 

Google Scholar 
111.Singh, K. I., Debbarma, A. & Singh, H. R. Field efficacy of certain microbial insecticides against Plutella xylostella Linnaeus and Pieris brassicae Linnaeus under cabbage-crop-ecosystem of Manipur. J. Biol. Control. 29, 194–202 (2015).Article 

Google Scholar 
112.Lin, H. P., Yang, X. J., Gao, Y. B. & Li, S. G. Pathogenicity of several fungal species on Spodoptera litura Chin. J. Appl. Ecol. 18, 937–940 (2007).
Google Scholar 
113.Martin, P. A. W., Hirose, E. & Aldrich, J. R. Toxicity of Chromobacterium subtsugae to Southern stink bug (Heteroptera: Pentatomidae) and corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 100, 680–684 (2007).PubMed 
Article 

Google Scholar 
114.Rodriguez, M. G. et al. Impact of entomopathogenic nematode applications on diamond back moth population. Rev. Protec. Vegetal. 28, 158–160 (2013).
Google Scholar 
115.Abbas, W., Javed, N., Haq, I. U. & Ahmed, S. Pathogenicity of Entomopathogenic nematodes against cabbage butterfly (Pieris brassicae) Linnaeus (Lepidoptera: Pieridae) in laboratory conditions. Int. J. Trop. Insect Sci. 1–7 (2020).116.Huang, Z., Ali, S., Ren, S. & Wu, J. Effect of Isaria fumosoroseus on mortality and fecundity of Bemisia tabaci and Plutella xylostella. Insect Sci. 17, 140–148 (2010).Article 

Google Scholar 
117.Kunimi, Y. Current status and prospects on microbial control in Japan. J. Invertebr. Pathol. 95(3), 181–186 (2007).PubMed 
Article 

Google Scholar 
118.Mohan, S., Raman, R. & Gaur, H.S. Foliar application of Photorhabdus luminescens, symbiotic bacteria from entomopathogenic nematode H. indica, to kill cabbage butterfly Pieris brassicae. Curr. Sci. 84, 1397 (2003).119.Baur, M. E., Kaya, H. K., Tabashnik, B. E. & Chilcutt, C. F. Suppression of diamondback moth (Lepidoptera: Plutellidae) with an entomopathogenic nematode (Rhabditida: Steinernematidae) and Bacillus thuringiensis Berliner. J. Econ. Entomol. 91(5), 1089–1095 (1998).CAS 
PubMed 
Article 

Google Scholar 
120.Sunanda, B. S., Jeyakumar, P. & Jacob, V. V. Bioefficacy of different formulations of entomopathogenic nematode Steinernema carpocapsae against Diamond back moth (Plutella xylostella) infesting Cabbage (Brassica oleracea var. capitata). J. Biopestic. 7, 210–215 (2014).CAS 

Google Scholar 
121.Razek, A. A. S. Pathogenic effects of Xenorhabdus nematophilus and Photorhabdus luminescens against pupae of the Diamondback moth Plutella xylostella. J. Pest Sci. 76, 108–111 (2003).
Google Scholar  More

The fossils described in the following section have been collected from two different layers presenting slightly different assemblages but with several overlapping taxa. The descriptions are presented here in a taxonomic order. However, plates have been kept separate for the two localities. Plates 1–3 present the assemblage of the UPL while plates 4–9 present the plants that were collected from the LPL.Incertae sedis Bryophyta

Sporogonites
37

Sporogonites sp. A
Fig. 3a–d; Fig. 7a–b

Figure 3(a–d) Sporogonites sp. A, (e–g) Sporogonites sp. B., (a) Specimen AM 7944. Scale = 2 cm. Gross view of specimen. Several parallel slender non branching axes terminated by elongate sporangia. (b) Specimen AM 7944. Scale = 5 mm. Detail of the top part of the plant showing the shape of one sporangium. (c) Specimen AM 7944. Scale = 5 mm. Detail of the top part of the plant showing the shape of one sporangium. (d) Specimen AM 7944. Scale = 1 cm. Detail of an isolated specimen. Shape of one sporangium is visible. (e) Specimen AM 7953. Scales = 1 cm. Gross view of specimen showing the non-branching slender axis bearing terminally an elongate sporangium. (f) Specimen AM 7953. Scales = 1 cm. Detail of the sporangium. (g) Specimen AM 7953. Scales = 5 mm. Detail of the distal part of the sporangium characterized by a small notch (at arrow).Full size image
MaterialThis plant is very abundant in some layers where the elongated stems cover the whole bedding plane. Individual stems are in most cases difficult to identify.DescriptionThe plant consists of bunches of elongated non-divided axes, each ending in one sporangium when complete (Figs. 3a–d, 7a–b). In places, dichotomies seem to occur, but they result from the superimposition of axes. Axes are arranged in parallel. They are 10–11.5 cm long and 0.9–1.3 mm wide. Width is constant along their entire length.The distal part of the stalk is marked by a progressive but clear flaring which identifies the position of the proximal part of the sporangium (Figs. 3b–d, 7b). Sporangia are 7–7.5 mm long and 3–3.5 mm wide.Sporangia are elongated in shape and were probably ovoid to ellipsoid before compression. Their distal part is rounded in outline. The surface of the sporangia is unclear due to the coarse nature of the preservation. In some specimens, a small notch is observed on either side, approaching the tip of the sporangia, defining a small hemispheric structure (see arrows on Figs. 3d, 7b).Identity and comparisonsThe occurrence of elongated sporangia borne singly at the top of smooth unbranched axes unambiguously points to the genus Sporogonites Halle2737. This genus is comprised of 4 (or 5) species: S. exuberans Halle37, S. chapmanii Lang and Cookson33, S. excellens Frenguelli29 and S. yunnanense Hsü10. An additional species was described by Gonez31 but was not validly published. Gonez31 named it Sporogonites punctatus in his unpublished PhD manuscript. However, according to the International Code of Nomenclature for algae, fungi and plants art. 30.9, this publication is not effective and hence not valid (op. cit., art. 32.1).All species chiefly differ in the shape and size of their sporangia. Sporogonites yunnanense presents notably small sporangia ranging from 3.2–4.5 mm in length and 1.4–1.8 mm in width. By contrast, S. excellens is characterized by generally big sporangia up to 5 mm in width and 7 mm in length that are borne on up to 5 mm wide stalks. Our specimens do not compare favourably to either of these two species. The occurrence of a rounded apex to the sporangia in our specimens exclude them from S. chapmanii which is characterized by pointed sporangia. The above described South African specimens conform in shape and size range of the sporangia to S. exuberans. However, similar sporangia were previously reported as Sporogonites sp. A by Gerrienne et al.2 and as a Sporogonites punctatus in Gonez31 from the Paraná basin (Brazil). Sporogonites exuberans and Sporogonites “punctatus” differ mainly by the occurrence in the latter of a minute conical ornamentation on the upper half of the sporangia. The Brazilian material is comparable in size and shape to the Impofu Dam material, however the nature of the preservation of the latter precludes determination of the presence or absence of the diagnostic sporangial ornamentation. In order to avoid misleading paleogeographic interpretations we therefore prefer to leave the taxonomy of this plant open.Age and distributionThe genus Sporogonites is a common component of the earliest floras. It is most frequently reported from Emsian aged deposits in which it constitutes a common and widespread taxon (for full list see31). Its oldest reported occurrences are from assemblages from the Late Silurian of Vietnam31. In the Lochkovian, it has thus far only been found in the Brazilian Ponta Grossa Formation2,31. It is noteworthy to mention that sporangial ornamentation aside, our material is comparable to this sole Lochkovian occurrence.Sporogonites

sp. B
Fig. 3e–g

MaterialOnly one specimen of this plant has been collected as a relatively well-preserved isolated stem.DescriptionThis plant consists of a long unbranched stem distally bearing a large elongated sporangium (Fig. 3e). The stem is straight and measures 66.0 mm long and 1.9–2.1 mm wide. The distal end of the stem is marked by a progressive widening corresponding to the beginning of the sporangium (Fig. 3f). From the point of widening to the tip, the sporangium measures 14.3 mm long and 6.1 mm wide. The sporangium reaches its maximum width after 9 mm (2/3 of total length). The sporangium is terminated by a hemispherical structure marked by a clear depression of the lateral outlines and demarcated by a line of denser mineralisation (see arrow on Fig. 3g). This structure is 3.4 mm wide and 2.1 mm high.Identity and comparisonAs for Sporogonites sp. A, the occurrence of an elongated sporangium borne singly on a smooth non branched axis suggests the genus Sporogonites Halle37. Lack of bifurcation cannot be unambiguously established as a result of the lack of preservation of the base. Nonetheless the size of the plant mitigates against other explanations. Moreover, the general shape of the sporangium conforms to the genus Sporogonites, being very similar to both Sporogonites exuberans and Sporogonites “punctatus”. This specimen is, however, much larger than any formally described Sporogonites species. Further taxonomic discussions are nonetheless deferred on account of the mediocre preservation of the single specimen.The presence in both this specimen and Sporogonites sp. A of a small hemispherical structure terminating the sporangium is significant. A similar structure was observed by Halle37 on the Sporogonites (Sporogonites exuberans) type material from Röragen. In the most complete specimens, the tip of the sporangia is described as rounded but with a little break differentiating darker material around the extremity of the sporangium. Alternately this structure may be absent, and the sporangial tip characterised by a depression. Cyrille Prestianni (CP) has confirmed presence of this structure in specimens of Sporogonites exuberans from Belgium. From the perspective of the common identification of Sporogonites, as a bryophyte, this recurring structure likely represents the operculum of the capsule.Polysporangiophyta

23

Cooksonia
32

Cooksonia paranensis2 .
Fig. 4a–n; Fig. 7c and f

Figure 4(a–n) Cooksonia paranensis2,4. (a) Specimen AM 7908. Scale = 1 cm. specimen showing three branching orders and a terminal sporangium. (b) Specimen AM 7906. Scale = 1 cm. Truss of the plant showing a number of branching orders and terminal sporangia. (c) Specimen AM 7910a. Scale = 5 mm. Sporangium. (d) Specimen AM 7887. Scale = 5 mm. Sporangium. (e) Specimen AM 7913. Scale = 5 mm. Sporangium. (f) Specimen AM 7912a. Scale = 5 mm. Sporangium. (g) Specimen AM 7888. Scale = 5 mm. Sporangium. (h) Specimen AM 7950b. Scale = 5 mm. Sporangium. (i) Specimen AM 7964. Scale = 5 mm. Sporangium. (j) Specimen AM 7958a. Scale = 5 mm. Sporangium. (k) Specimen AM 7965. Scale = 5 mm. Sporangium. (l) Specimen AM 7955a. Scale = 5 mm. Sporangium. (m) Specimen AM 7959a. Scale = 5 mm. Sporangium. (n) Specimen AM 7954b. Scale = 5 mm. Sporangium.Full size image
MaterialThis plant is the most abundant in the UPL. It occurs either as isolated sporangia, sporangia connected to small stem fragments or as bunches of fertile axes. By contrast only two specimens have been identified in the LPL.DescriptionSeveral specimens of this plant have been discovered (Figs. 4, 7c,f). They consist of isotomously branched axes, 0.7–1.2 mm in width. In many cases, it is difficult to distinguish individual branching systems as, when not fragmentary, plants occur in bunches (Fig. 4b). This is particularly the case on one specimen that shows several isotomously branched plants arising from the same point (Fig. 4b). The specimens in Fig. 4a,b show the ultimate branching orders with sporangia attached. Branching systems always bear terminal sporangia (Fig. 4). Sporangia are trumpet- to cup-shape in outline and measure 2.5–5.0 mm in diameter and 2.0–3.0 mm in height. The axis/sporangium transition is progressive (Fig. 4c–m). It is thus difficult to identify with precision the base of the sporangium. The sporangial cavity gives the impression of being sunken into the subtending axis (Fig. 4f–k). The upper part of the sporangium is flat and marked by the presence of an apical plateau. The shape of the apical plateau seems very variable, but it results from differences in compression orientation (Fig. 5). In most cases, it is folded up, which results in a bulge (Figs. 5a, 4c–e) or in wrinkles (Figs. 5b, 4g). Several specimens seem to present a more spherical structure rather than an apical plateau (Fig. 4f,j–n). The apparent rounded shape of the sporangia is the result of the tilting of the apical plateau (Fig. 5c). This configuration was already noted by Gonez and Gerrienne7.Figure 5Schematic reconstruction of the sporangia of Cooksonia paranensis showing different position of the operculum (op) in regard to the sporangial chamber (sc): (a) in growth position with the operculum in place, (b) with the operculum compressed laterally showing several wrinkles and (c) with operculum completely tilted.Full size imageIdentity and comparisonsPlants with smooth isotomously branched axes, gradually widening distally into a single, terminal, cup- or trumpet shaped sporangium with an apical plateau can be attributed either to the genus Cooksonia Lang emend6 or to the genus Concavatheca52.The genus Cooksonia was originally described by Lang32 on the basis of compression fossils exhibiting “dichotomously branched, slender, leafless stems, with terminal sporangia that are short and wide [with an] epidermis composed of elongate, pointed, thick-walled cells [and a] central vascular cylinder consisting of annular tracheids”. Thanks to the works of Edwards and collaborators (see among others:35,36,7,9), the genus is now known in great detail, on the basis of both compression and coalified specimens. The type species C. pertoni is considered the earliest eutracheophyte23. The genus diagnosis was emended in 20106 at which time it included three well defined species: C. pertoni, C. paranensis and C. banksii6, however C. banksii has later been transferred to another genus (see below and Morris et al.52). C. pertoni and C. paranensis are morphologically similar, but, according to Gonez and Gerrienne6, C. paranensis can be distinguished from C. pertoni by its slender axes and the more gradual transition between axis and sporangium. As a result of this gradual transition, the sporangial cavity of C. paranensis is sunken in the subtending axis. The genus Cooksonia also includes three less well-preserved species, C. hemisphaerica32, C. cambrensis35 and C. bohemica38. All of these are considered doubtful by Gonez and Gerrienne6 because they are based on poorly preserved specimens. A restudy of the fossil material of C. bohemica has led Kraft et al.41 to place this plant within the genus Aberlemnia under the combination A. bohemica. Recently, a new species, C. barrandei64 has been described. The plant is morphologically close to C. pertoni and C. paranensis, but with more robust axes and bigger sporangia64.The specimens originally described under the binomial Cooksonia banksii by Habgood et al.7 have been transferred to the genus Concavatheca by Morris et al.52. The genus Concavatheca includes plants with smooth axes and single terminal sporangia. The subtending axis gradually widens distally into a cup-shaped, sunken sporangial cavity. The specimens of Concavatheca banksii differ from those of Cooksonia. pertoni because the spore mass of Concavatheca banksii is sunken in the subtending axis whereas Cooksonia pertoni has a discoidal spore mass subtended by an axis that gradually increases in diameter. Other differences exist, both in sporangial structure and spore ultrastructure. In having a sunken sporangial cavity, the species Concavatheca banksii is very similar to Cooksonia paranensis and the two species are difficult to distinguish. They are mainly differentiated by their preservation type: compression for C. paranensis and charcoalification for Concavatheca banksii. Accordingly, detailed comparisons are not possible, and the two species can be kept in separate genera, on the basis of art. 11.1 of the International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code)18, which states that “the use of separate names is allowed for fossil-taxa that represent different parts, life-history stages, or preservational states of what may have been a single organismal taxon or even a single individual”18, art. 11.1).The specimens here described are morphologically very close to Cooksonia pertoni, Cooksonia paranensis and Concavatheca banksii. The very gradual transition between the sporangia and the subtending axes as well as the “sunken” aspect of the sporangial cavity are reminiscent of both C. paranensis and Concavatheca banksii. As our material, like previously described material of Cooksonia paranensis, is preserved as compression fossils it cannot be accurately compared with Concavatheca banksia, and we accordingly choose to name it Cooksonia paranensis.Age and distributionCooksonia paranensis was first described from the Ponta Grossa Formation at Jackson de Figueiredo (southern Paraná Basin, Brazil) as well as in four other localities of the Paraná Basin2. A Lochkovian age has been proposed for these plant bearing beds based on palynology2. One single other putative occurrence has been recorded. In the Lochkovian Talacasto Formation, at Talacasto creek (Argentina) Edwards et al.43 report one poorly preserved specimen that could be assigned with doubts to either Cooksonia paranensis or Concavatheca banksii.
Cooksonia hemisphaerica32
Fig. 6a–b.

Figure 6(a,b) Cooksonia hemisphaerica, (c) Cooksonia cambrensis Edwards35, (d–f) Tortillicaulis sp., (g–h) cf. Cooksonia hemisphaerica, (a) Specimen AM 7914. Scale = 1 cm. Specimen showing the gross morphology of the plant. (b) Specimen AM 7914. Scale = 5 mm. Detail of a rounded sporangium that is clearly distinct from the widening subtending axis. (c) Specimen AM5828. Scale = 2 mm. Sporangium and subtending axis. (d) Specimen AM 7956a. Scale = 5 mm. Specimen showing the organisation of the sporangia. An oblique striation is visible at the surface of the sporangium. (e) Specimen AM 7943. Scale = 5 mm. Specimen showing naked axes bifurcating one time before terminating in elongate sporangia. Note the oblique striation at the surface of the sporangia. (f) Specimen AM 7921a. Scale = 5 mm. A dichotomizing naked axis terminally bearing two sporangia. The sporangia bear a distinct oblique ornamentation and are made of two halves that are twisting on each other. (g) Specimen AM 7915a. Scale bar = 1 cm. Specimen showing the gross morphology of the plant. (h) Specimen AM 7925. Scale bar = 5 mm. Detail of specimen Fig. 6g showing the organisation of the sporangia.Full size image
Figure 7(a,b) Sporogonites sp. A, (c) and (f) Cooksonia paranensis, (d–e) and (g–h) Steganotheca striata. (a) Specimen AM 7927. Scale = 1 cm. A truss of non-branching more or less parallelly oriented axes distally bearing one single sporangium. (b) Specimen AM 7927. Scale = 5 mm. Detail of the distal ends and of the sporangia. The arrow point towards the limits of the small hemispheric structure present at the tip of the sporangium. (c) Specimen AM 7891a. Scale = 1 cm. Gross morphology of the specimen showing several superimposed individuals. (d) Specimen AM 7893. Scale = 1 cm. Gross morphology of the specimen showing several intertwined axes. (e) Specimen AM 7997. Scale = 5 mm. Isolated termination showing the ultimate dichotomy and two sporangia. (f) Specimen AM 7886. Scale = 5 mm. Detail of an isolated sporangium. (g) Specimen AM 7987. Scale = 5 mm. Distal part of the plant showing two more or less parallel sided sporangia. (h) Specimen AM 7998. Scale = 5 mm. Distal part of the plant showing two more or less parallel sided sporangia. (i) Specimen AM 7973. Scale = 5 mm. Isolated specimen showing the organisation of the sporangium.Full size image
MaterialOne single moderately preserved specimen from the UPL.DescriptionThe specimen consists of a 58 mm long dichotomous branching system terminally bearing sporangia (Fig. 6a). Two isotomous dichotomies are observed (see arrows on Fig. 6a). The first order axis is broken at its base and measures 20 mm long and 1 mm wide. Second order axes are 10–12.5 mm long and 0.8–1 mm wide. Finally, third order axes are short and measure 5 mm long and 1 mm wide.Sporangia are globular, they measure between 1.3–2.0 mm long and 2.0–2.4 mm wide. Within the sporangium, a circular structure can be observed that we interpret as the sporangial cavity (Fig. 6b). This cavity measures between 1.5 and 1.8 mm in diameter. Sporangial wall is 0.3–0.4 mm thick. No dehiscence line could be observed.The subtending axis is 2.9–3.6 mm long. It gradually flares and distally reaches 2.0–2.4 mm in width. The sporangium-axis contact is relatively large as the axis is almost as wide as the sporangium (Fig. 6b).Identity and comparisonDichotomous branching systems distally bearing elongate structures are relatively common in the Lower Devonian. Identification is made difficult by the scarcity of morphological traits. In addition to Cooksonia hemispherica, four taxa are known to show such organisation. They are: Tortilicaulis Edwards35, Tarrantia Fanning et al.36, Salopella Edwards and Richardson44 and Uskiella Shute and Edwards12. They however all present an elongated sporangial cavity and a sharp transition at the sporangium-axis contact. The occurrence of isotomously branched axes distally bearing globular sporangia at the end of gently tapering subtending axes conforms to Cooksonia hemisphaerica Lang32,35,36. The here recorded size range conforms to the larger forms recorded from the British Isles.Age and distribution. Cooksonia hemisphaerica was originally described from the Targrove quarry32. The Targrove quarry deposits have been dated by means of fishes and spores and are Lochkovian in age36. Later, Edwards35 reported specimens of Cooksonia hemisphaerica from Freshwater East in the South Dyfed region (South Wales). This occurrence has been dated through palynology and is considered Pridoli in age. Another occurrence is the Lochkovian Brown Clee Hill locality (Shropshire-England)46. Cooksonia hemisphaerica has also been recorded in the Bryn Glas borehole (Anglo-Welsh Basin)42.
Cf. Cooksonia hemisphaerica32
Fig. 6g–h
MaterialThis plant is only known from a single more or less complete specimen collected from UPL.DescriptionIt is characterized by a dichotomous branching system distally bearing globular sporangia (Fig. 6g–h). It is 130 mm long and consists of a three-times isotomously dichotomizing axis. The branching angles are small measuring less than 10°. First order axis is proximally incomplete and measures 35 mm long and 1.9 mm in width. Second order axes are 49 mm in length and 1.0 mm in width. The third order axes are 25–27 mm in length and 0.8–0.9 mm in width. The fourth ultimate order axes are 16–19 mm in length and 0.6–0.8 mm in width. Each third order axis bears one sporangium. The subtending axes of the sporangia gradually flare at a length of 6.0–7.0 mm to approximately reach the width of the sporangia (Fig. 6h). The sporangia are globular and measure 1.0–1.5 mm long. When measured at their widest point, sporangia are 2.0–2.2 mm in width.Identity and comparisonsDespite notable differences, the shape of the Impofu Dam specimens is most reminiscent of Cooksonia hemisphaerica. In this plant, sporangia are globose and borne on axes strongly widening just below them32,36. The recorded size range for the sporangia in C. hemisphaerica is compatible with the Impofu Dam material. However, C. hemisphaerica differs in that its sporangia are produced shortly after the ultimate dichotomy. Moreover, the identity of the “rounded tip” as a sporangium is equivocal as no clear division from the axis is visible. Therefore, we cannot unequivocally assign this material to C. hemisphaerica and so name the fossil cf. Cooksonia hemisphaerica.
Cooksonia cambrensis35
Fig. 6c
MaterialOne single isolated sporangium from the UPL.DescriptionSporangium born singly at the end of an unbranched smooth axis (Fig. 6c). The axis is 18 mm long and 0.7 mm wide. It slightly tappers distally to reach 1 mm at the base of the sporangium. The sporangium-axis junction is clear and flat. The sporangium is elliptical in outline, but we here suspect some deformation to have occurred. It measures 2.8 mm wide and 1.4 mm high.Identity and comparisonAlthough occurring as a single isolated specimen, it remarkably conforms to Cooksonia cambrensis to which we assign it based on the shape of the sporangium and the very limited tapering of the subtending axis35,36.Age and distributionCooksonia cambrensis has been identified in the Pridoli of Wales35,47 and the Lochkovian of England36,47.
Steganotheca48
Steganotheca striata48
Fig. 7d–e; 7 g–h.
MaterialThis plant has exclusively been found in the LPL. Four specimens have been recovered occurring either as isolated stem fragments or as a relatively densely occurring plant mat.DescriptionOnly the ultimate one or two branching orders have been recovered, up to 35 mm in overall length. The axes are isotomously branched (Fig. 7d–e, g–h). Their width is relatively constant throughout specimens and range from 0.6–1.4 basally to 0.8–1.4 mm distally. The axial surface is smooth. The sporangia are borne singly. They measure 2.9–4.2 mm long and 1.9–3.0 mm wide. The subtending axis widens rapidly at the transition to the sporangium. The width of the sporangium then remains constant for most of its length. The tip of the sporangium is truncated and seems to be topped by a denser looking lens-shaped apical plateau. The whole structure has the shape of a mug.Identity and comparisonThe occurrence of smooth isotomous branching systems bearing isolated sporangia characterized by a flaring of the axes and topped by an apical plateau is indicative of either the genera Cooksonia32 or Steganotheca48. They chiefly differ in the shape of the sporangia. The occurrence of mug-shape terminal sporangia, that are longer than wide, parallel sided and truncated at the apex conforms to the genus Steganotheca. Despite the lack of minute details such as the striation observed on the sporangia of the original material from South Wales, we consider the similarities sufficient to attribute the Impofu dam material to the species Steganotheca striata.Age and distributionSteganotheca striata has been recorded in the Silurian (Ludfordian and Pridoli) of South Wales (Capel Horeb Quarry)48,49.
Aberlemnia5
Aberlemnia caledonica (Edwards)5
Fig. 8a–d

Figure 8(a–d) Aberlemnia caledonica, (e) Tortilicaulis sp., (f–h) Uskiella spargens. (a) Specimen AM 7970. Scale = 1 cm. Gross morphology of the plant showing its dense organisation. (b) Specimen AM 7970. Scale = 5 mm. Detail showing the organisation of the sporangia. (c) Specimen AM 7985. Scale = 5 mm. Detail of a subcircular sporangium. (d) Specimen AM 7980. Scale = 5 mm. Detail several subcircular sporangia. (e) Specimen AM 7984a. Scale = 5 mm. Ultimate dichotomy of the plant bearing two sporangia. Note the occurrence of an oblique striation at the surface of the sporangia. (f) Specimen AM 7966a. Scale = 1 cm. Isolated specimen showing the gross morphology of the plant. Note the horizontal axis and the marked curvature. (g) Specimen AM 7968a. Scale = 1 cm. Specimen showing the gross morphology of the plant. (h) Detail of specimen in fig. 7f showing two sporangia. Scale = 0.5 cm.Full size image
MaterialFour specimens including a more or less complete branching system have been recovered from the LPL.DescriptionThe largest specimen of this plant is figured in Fig. 8a. It is relatively densely packed and therefore it is difficult to describe individual branching systems. It consists of branched axes terminated by reniform to transversely elongated sporangia (Fig. 8a,b). The axes are smooth and mostly dichotomize isotomously. They are, however, some indications of anisotomous division, but preservational limitations precludes any definite statement thereon (Fig. 8a). Axial width is constant throughout specimens and measure 0.5–0.7 mm. Branch length decreases distally. The ultimate division gives rise to short axes. Sporangia are sub-circular in outline and measure 1.0–1.8 mm in width and 0.9–1.6 mm in length (Fig. 8b–d). Subtending axes widen sharply just beneath sporangia and reveal a curved axis-sporangium junction.Identity and comparisonThe occurrence of sub-circular terminal sporangia on mostly isotomously dichotomizing axes points towards the genera Aberlemnia and Sporathylacium. The prior was erected by Gonez and Gerrienne5 in order to accommodate specimens with reniform sporangia formerly included in the genus Cooksonia. The latter was established by Edwards et al.50 for bivalved reniform anatomically preserved sporangia. In the absence of any evidence for bivalved sporangia and considering the lack of anatomical details and of in situ spores in the Impofu dam material, we assign this material to Aberlemnia caledonica.Age and distributionAberlemnia has been recorded from the Lochkovian of Scotland48, the late Silurian to Lochkovian of Wales35,36,47,49, the Lochkovian of Brazil2, and possibly from the Ludlow of Bolivia51,74. Recently, Kraft et al.41 have suggested that the Late Silurian Cooksonia bohemica should be reassigned to the genus Aberlemnia. However, further studies are necessary to ascertain validity of this proposition.
Tortilicaulis35
Tortilicaulis sp.
Fig. 6d–f; Fig. 8e;
MaterialSpecimens of this plant consists of 3 specimens from the UPL and one specimen from the LPL.DescriptionIn all cases only one (Fig. 6d) or two (Figs. 6e–f, 8e) ultimate branching orders have been preserved. The preserved plant comprises large elongate sporangia terminating dichotomous axes. Sporangia are fusiform and taper distally to a blunt tip. The tip is obscured in specimen Fig. 6d by the occurrence of a transversely oriented axis. The sporangia show a clear twisted organization (see arrows on Fig. 6e,f). An obliquely oriented striation is observable on the surface of most sporangia. The measured angle of this striation is relatively variable and measures 24° (Fig. 6e), 28° (Fig. 6d) and 38° (Fig. 8e) from the vertical. A similarly oriented longitudinal splitting here interpreted as a dehiscence line is repeatedly observed (Figs. 6e–f, 8e). This is particularly visible on specimen Fig. 6f. This specimen is dehisced, and the putative two valves of the sporangia are slightly separated and twisted around each other. The junction with the subtending axis is unclear except on Fig. 6f where it is marked by the beginning of the putative dehiscence line. Maximum width of the sporangia occurs at mid-height. They measure 1.2–1.4 mm long and 0.3–0.4 mm wide in the UPL and 0.6 mm long and 0.2 mm wide in the LPL. The height to width ratio ranges between 3 and 3.5. The subtending axes are parallel sided but a faint widening upwards can be seen. Obliquely oriented cellular patterns could be observed on the subtending axes. Dichotomies are largely isotomous, branching at an acute angle between 10° and 15°.Identity and comparisonThe occurrence of relatively large fusiform sporangia terminating isodichotomously branched smooth axes with relatively low branching angles, points towards three genera, namely Salopella Edwards and Richardson44, Tortilicaulis35 and Teruelia53. Salopella is characterized by longitudinally aligned elongated cells on the sporangia whereas these are obliquely oriented in Tortilicaulis and Teruelia. The latter however differs in being characterized by a multi-slit dehiscence whereas Tortilicaulis exhibits only one dehiscence slit. The oblique striation and the single dehiscence slit observed on the Impofu dam sporangia therefore support assignment to the genus Tortilicaulis. The only two species included in this genus are T. transwalliensis and T. offaeus. The Impofu dam material all falls within the size range of T. transwalliensis as described from the Targrove quarry36. The height to width ratio is however slightly smaller in the South African material but falls within the range of T. transwalliensis in general35,36. Tortilicaulis transwalliensis includes a very large size range from very small to relatively large forms. Size and proportion of the sporangia therefore appear to provide a relatively weak taxonomic guideline. Besides the characteristics of the sporangia, the main difference between the Impofu dam material and previously described T. transwalliensis is the presence of a very long subtending axis. Although, morphologically very close to T. transwalliensis we cannot unequivocally assign our new material to this species and so assign the fossil to Tortilicaulis sp.Age and distributionThe genus Tortilicaulis was first described from the Pridoli of South Wales35. The species T. transwalliensis has further been reported from the Ludlow Targrove Quarry (Shropshire, UK)36 and from the Lochkovian Bryn Glas Borehole (South Wale, UK)42. Tortilicaulis offaeus has been described from the Lochkovian of North Brown Clee Hill locality (Shropshire, UK)46. Tortilicaulis cf. offaeus from the Lochkovian Tredomen Quarry (South Wales, UK). Another hypothetical record comes from the Lower Devonian (Lochkovian?) Argentinian Villavicencio Formation with the record of a cf. Tortilicaulis54.
Uskiella12
Uskiella spargens12
Fig. 8f–h
MaterialSeven specimens were recovered from the LPL.DescriptionThis plant consists of isodichotomous naked axes terminated by ovate structures. We assume these structures to be sporangia even though no in situ spores were observed.One exceptionally large specimen, AM7968, is 51 mm long and consists of an axis that branches six-times (Fig. 8g). The first order axis is broken and measures 1.3 mm in diameter. It is considered to have been horizontal. After the first dichotomy, it gives rise to two axes that curve upwards 6 and 7 mm following an angle of 91° and 94°. They both measure 14 mm long and 1.3 mm wide. Each of these further dichotomizes at most three more times. The branching pattern is obscured by the density of the branching. All divisions appear to occur isodichotomously even though a slight anisotomy is possible. Third order axes are 9–13 mm long and 0.7–0.8 mm wide; fourth order axes are unclear but measure between 3 and 7 mm long and 0.6–0.8 mm wide; fifth order axes are 5–8 mm long and 0.6–0.8 mm wide. The sixth order axes constitute the ultimate one and each terminate in a sporangium. These are very short and measure 1.2–3.5 mm long and 0.6–0.8 mm wide. The gradual axis/sporangium transition makes precise measurement of the sporangium and its subtending axis difficult. Thus, we arbitrarily considered the place where the subtending axis is no longer parallel sided to mark the base of the sporangium. The sporangia are ovate in shape. They are characterized by a widening of the subtending axis that gives to the whole structure the shape of a tennis racket. They measure 3.4–4.7 mm long and 1.3–2.3 mm wide. No specialized structure for dehiscence is observed. The outline of the sporangia is very variable. The axis/sporangium junction is difficult to observe with precision but is marked by a slightly convex to almost straight line.Another large specimen is AM7966 (Fig. 8f). It is 45 mm long and consists of an axis that branches three-times. The first order axis is 25 mm long and is presumed to have been horizontal for the first 14 mm, after which it is inflected by 108°. It measures 1.2 mm wide before the inflection and 1.4 thereafter. The plant subsequently dichotomizes three times. Second order axes are 9.5–10.8 mm long and 12–1.3 mm wide; third order axes are 5.4–6.8 mm long and 0.9–1.0 mm wide; fourth order axes are 4.1–5.9 mm long and 0.7–0.8 mm wide. The ultimate order axes measure 1.8–2.5 mm long and 1.0–1.2 mm wide. Only two sporangia are sufficiently well preserved to be accurately studied (Fig. 8h). They are rounded to ovate in shape and measure 6.2 and 3.4 mm long and 2.9 and 3.9 mm wide respectively. As with the previous specimen the axis/sporangium junction is marked by a slightly convex to almost straight line.In summary, this plant seems to be characterized by a horizontal axis that dichotomizes at least once before curving at an angle approaching 90° and giving rise to what we interpret as the erect part of the plant. It subsequently dichotomizes three more times. Dichotomies seem to occur more or less isotomously but branching is relatively difficult to interpret in both specimens. The sporangia are tennis-racket shaped in outline..Identity and comparisonsThe presence of longitudinally elongated sporangia showing a tapering base is initially suggestive of the genus Salopella Edwards and Richardson44. However, this genus differs significantly in shape as it is consistently described as having sporangia with acute tips and tapering apices44,46.Rounded to elongate relatively large sporangia have repeatedly been reported in the literature56. The shape of these sporangia resembles two specimens originally described by Croft and Lang56 as Cooksonia sp. but later identified as cf. Sporogonites by Cookson57. They were both rediscussed by Shute and Edwards who highlighted the occurrence of a longitudinal slit on the sporangia which defined two valves. The material was therefore reassigned to the species Uskiella spargens. We believe that the Impofu Dam material is more or less identical to adpression material thereof from both Wales (UK) and Victoria (Australia). Evidence for the occurrence of two valves in our material is tenuous, however a double valved sporangium would explain the variability of the shape of the sporangia observed in several specimens (Fig. 8h). In addition, the Impofu Dam specimens fall within the same size range as Uskiella spargens. Uskiella reticulata36 is characterized by much smaller sporangia. Considering the many similarities existing between the Impofu Dam material and both the Australian and Welsh material and despite the lack of definite evidence of a longitudinal dehiscence slit, we attribute the here described material to Uskiella spargens.RemarksOne of the important features of this plant is the description of an extensive branching system. The occurrence of a dichotomizing horizontal axis giving rise to an erect plant has repeatedly been observed in plants of more or less the same age such as Aglaophyton majus, Rhynia Gwynne-vaughannii or Nothia aphylla (see Hetherington and Dolan for references). The lack of anatomical preservation in the Impofu Dam material precludes a rigorous interpretation of these axes. It is, however, hypothesised that these dichotomizing horizontal axes performed the sporophyte rooting function. This suggest that Uskiella spargens lacked a true rooting system.Age and distributionUskiella spargens was originally described from the Pragian of Wales12 Specimens identified as Cooksonia sp. by Croft and Lang56 and later synonymized with U. spargens were collected from the Lochkovian of Allt Du (South Wales)59. Specimens originally described as cf. Sporogonites but synonymized with U. spargens by Shute and Edwards12 were collected from the Lochkovian to lower Pragian Humevale Siltstone Formation of Lilydale (Australia)57.GenusKrommia gen. nov.Type species: Krommia parvapilla sp. nov.Derivation of the name: Krommia from the Kromme River (from Afrikaans meaning curved).Diagnosis: Plant with smooth, three dimensional, isotomously branching axes; Sporangia small and rounded borne singly and terminally.SpeciesKrommia parvapila sp. nov.Derivation of the name: parvapila, from Latin a small ball referring to the sporangium.Diagnosis: Same as for genus. Dichotomizing up to three times. Branching angle variable (40°–110°). First and second order axes U shaped. Axes 1.5–3.0 mm long and 0.3–0.35 mm wide below sporangia. Small constriction at junction between subtending axis and sporangium. Sporangia rounded between 0.7 and 0.8 mm in diameter.Holotype: AM 7928a (part) and AM 7928b (counterpart), Fig. 9a.Figure 9(a–c) Krommia parvapilla gen. nov. sp. nov., (d–l) Elandia itshoba gen. nov. sp. nov. (a) Specimen AM 7929. Scale = 5 mm. Gross morphology of the plant showing the U-shaped first and second order axes. (b) Specimen AM 7928a. Scale = 5 mm. Slightly laterally compressed specimen showing the different branching orders and the terminal rounded sporangia. (c) Specimen AM 7969b. Scale = 5 mm. Specimen the U-shaped first order axes and the terminal sporangia. (d) Specimen AM 7894a. Scale = 5 mm. (e) Specimen AM 7897a. Scale = 5 mm. (f) Specimen AM 7975a. Scale = 5 mm. (g) Specimen AM 7988a. Scale = 5 mm. (h) Specimen AM 7975b. Scale = 3 mm (i) Specimen AM 7988a. Scale =  4 mm(j) Specimen AM 7932a. Scale = 5 mm. (k) Specimen AM 7931a. Scale = 3mm (l) Specimen AM 7975a. Scale = 3 mm.Full size imageParatypes: AM 7929 and AM 7969.Repository: Albany Museum, Devonian Lab, Beaufort Street, Makhanda, Eastern Cape, South Africa.Type locality: Impofu Dam, Kouga Municipality, Eastern Cape, South Africa (Fig. 1).Horizon: Kareedouw Member, Baviaanskloof Formation, Nardouw Subgroup, Table Mountain Group, Cape Supergroup.Age: Lower Devonian, Lochkovian?Synonymy: Minutia fragilis nomen nullum Gonez31, Fig. 1 p. 178, from Jackson de Figueiredo, Jaguariaiva county, Brazil.
Fig. 9a–c
MaterialFive specimens of this plant have been recovered from the LPL.DescriptionIn all specimens, only the ultimate parts of the plants have been preserved. They measure up to 20 mm in length and consist of smooth axes that branch isotomously at relatively variable angles (40–110°). This variability suggests that the branching system was three dimensional. We think that relatively wide angles (70–110°) were originally present and compressed during taphonomical processes. A maximum of three dichotomies has been observed however the occurrence on Fig. 9a of two superimposed similar branching systems in the same orientation suggests that more dichotomies can be expected. On this specimen only a small part of the first branching order has been preserved which measures 0.9 mm wide. It branches to produce two slightly different axes measuring 8–10 mm long and 0.6 and 0.7 mm wide respectively. The exact branching pattern is then obscured by the superimposition of at least one other branching system; however, it clearly branches two more times. The first and second branching orders are characterized by a slight curvature that give to the pair of axes a U rather than a V shape. The specimens are terminated by small rounded sporangia measuring 0.7–0.8 mm wide. The junction between the subtending axes and the sporangia is marked by a small constriction of the axis. Subtending axes measure 1.5–3.0 mm long and 0.3–0.35 mm wide.A similar branching pattern is observed in specimens illustrated in Fig. 9b and c. First axes orders are incomplete and measure 6.5–7.5 mm long and 0.5–0.8 mm wide respectively. Second branching orders measure 3.8–4.8 mm long and 0.3–0.4 mm wide in Fig. 9b and 5.0–8.0 mm long and 0.6–0.7 mm wide in Fig. 9c. Third order branches measure 2.0–3.5 mm long and 0.3 and 0.5 mm wide in Fig. 9b and 2.5–3.5 mm long and 0.4–0.6 mm wide in Fig. 9c. The fourth order axes also are the subtending axes of the sporangia. They measure 2.4–2.8 mm long and 0.3–0.5 mm wide in Fig. 9b and 3.4–3.5 mm long and 0.3–0.4 mm wide in Fig. 9c. They always end in a small constriction that marks the base of the sporangia. The sporangia are rounded and measure 0.7–0.8 mm in diameter. No dehiscence feature was observed.ComparisonSmall plant remains with minute (mesofossil sized) sporangia have repeatedly been observed in Silurian to Lochkovian deposits60,62,11,46,73. Occurring as isolated plant fragments most of them were kept in open taxonomy. Several plant fragments resembling the Impofu Dam material were illustrated by Morris et al. 73. Our specimens most closely resemble their morphotype C however the characteristic constriction at the base of the sporangia has not been reported. One of their illustrated specimens does however show a similar structure. Croft and Lang56 published several specimens as Cooksonia sp.. Mainly consisting of isolated sporangia, they could bear some superficial resemblance to the SA specimens however detailed comparison is made difficult by the absence of vegetative structures. Our specimens more closely resemble the more fragmentary Brazilian specimens illustrated in the unpublished thesis of Gonez31. They share the same overall organization including the curvature of the second and third order axes. The characteristic constriction at the base of the sporangia is also present and the sporangia are comparable in shape and size, suggesting that they represent the same species. The Brazilian material has however never been validly published. Considering the extensive branching system preserved and the occurrence of this plant both in Brazil and South Africa we chose to erect a new genus and species.GenusElandia gen. nov.Type species: Elandia itshoba sp. nov.Derivation of the name: after Eland (Taurotragus oryx), from Elandsjacht, original farm name of locality, meaning Eland hunt in Afrikaans.Diagnosis: Plant forming dense trusses of fine, smooth, isotomously branching axes. Axes bifurcating at a low angle and terminating in minute, elongate ovate sporangia; multiple axes united by a basal structure.SpeciesElandia itshoba sp. nov.Derivation of the name: from isiXhosa, itshoba, a ritual fly whisk made from a bulls tail, sometimes with the hair tips decorated with tiny beads.Diagnosis: As for genus, bifurcating up to four times, sporangia straight sided with broadly rounded apices. Sporangia 1.3–1.6 mm long and 0.4–0.7 mm wide.Holotype: AM 7932a (part) and AM 7932b (counterpart), Fig. 9j.Paratype: AM 7894, AM 7897, AM 7975, AM 7988.Repository: Albany Museum, Devonian Lab, Beaufort Street, Makhanda, Eastern Cape, South Africa.Type locality: Impofu Dam, Kouga Municipality, Eastern Cape, South Africa (Fig. 1).Horizon: Kareedouw Member, Baviaanskloof Formation, Nardouw Subgroup, Table Mountain Group, Cape Supergroup.Age: Lower Devonian, Lochkovian?
Fig. 9d–l
MaterialSeven specimens from the LPL.DescriptionThis plant most often occurs as densely packed trusses of axes. This renders description of individual axes’ organization difficult. In all specimens, it consists of up to 60 mm long thin smooth axes that branch up to four times and bear minute terminally elongate ovate sporangia.The organization of the plant is best seen in AM 7932a (Fig. 9j), which is proximally incomplete. It measures 33 mm long and branches 4 times. 7.2 mm of the first order axis is preserved which is 0.3 mm wide. Second order axes are 7.0 and 9.5 mm long and 0.2 and 0.3 mm wide respectively. Fourth order axes are 6.5–8 mm long and 0.2–0.3 mm wide. Only one fifth order axis is preserved and measure 4.5 mm long and 0.3 mm wide. The sixth order axes correspond to the subtending axes of the sporangia. They measure 2.4–3.1 mm long and 0.2–0.3 mm wide. The junction between the sporangium and the subtending axis is clear.The base of the plant is best seen on Fig. 9f,g. When preserved, first order axes are long, only dichotomize after 18 to 19 mm and are 0.3 mm wide. Several more or less parallelly disposed axes converge basally on a poorly preserved structure of indefinite shape (Fig. 9h,l). Up to 6 axes are observed to arise from this structure that is here interpreted as a remnant of the gametophyte. It is up to 4 mm wide. Figure 9d and e however suggest that in life a larger number of axes were probably attached to a more extensive gametophyte or cluster of gametophytes.The sporangia are vertically elongated and straight sided with broadly rounded apices. They measure 1.3–1.6 mm long and 0.4–0.7 mm wide.Identity and comparisonAs discussed for Krommia parvapilla, the occurrence of very small sporangia has been reported many times, mostly from Wales. In the majority of cases however, they are found isolated or connected to very fragmentary branching systems. The very limited available information (mostly the shape of the sporangium) makes comparison and identification not only difficult but also very likely misleading. The extensive branching system preserved in the Impofu Dam material allows for a better description of the plant to be made. Three features are noticeable, the shape of the sporangia (elongated, parallel sided and rounded tips), the delicate branching system with relatively small branching angles and the occurrence of possible gametophytic tissues at the base of the plant. As already discussed above, even if the shape of the sporangia is by far the most informative character in early land plants, it is also misleading as they are very simple. When comparing the sporangia alone, the Impofu Dam material most closely resembles Tarrantia salopensis Fanning et al.36 and Uskiella reticulata Fanning et al.36. The sporangia are however distinct, being much smaller and presenting a height/width ratio of 3.3 which is much higher than that encountered in these two species. The sporangia further lack the characteristic reticulation of U. reticulata. When comparing the whole plant, the Impofu Dam material very closely resembles Eogaspesia gracilis Daber75. This plant has been described as small slender dichotomizing axes being borne on a thicker dichotomous rhizome and bearing small ovate sporangia. Based on the illustrations, the connection between the axes and the so-called rhizome are dubious. Despite superficial resemblance, the south African material differs from Eogaspesia by being smaller (80–90 mm long for Eogaspesia as opposed to 60 mm for Elandia) and by presenting isotomous divisions only. The sporangia though very simple and thus difficult to compare differ in shape being more elongate (length/width ration of 3.8 in Elandia as opposed to 2.5 in Eogaspesia) are more parallel sided and have more rounded tips. We therefore exclude the Impofu Dam material from these two taxa. As far as we know, the combination of characters described above has never been encountered before. We thus chose to erect a new genus and a new species.GenusMtshaelo gen. nov.Type species: Mtshaelo kougaensis sp. nov.Derivation of the name: isiXhosa, a traditional broom.Diagnosis. Plant with multiple (at least 6) elongate sporangia, spindle-shaped in profile and evenly tapering to acute terminations, truncated proximally at point of attachment; arranged in a truss of sporangia that terminates elongate parallel sided isotomously bifurcating axes.SpeciesMtshaelo kougaensis sp. nov.Diagnosis. As for genus, robust axes bifurcate at least twice and widen slightly towards the terminal truss of sporangia; individual sporangia 0.74 to 0.8 mm wide and 4–6 mm long with a longitudinal dehiscence line. Vegetative axes 1.0 to 1.4 mm wide.Derivation of the name: Kouga is the name of the district in which the site is found, from Khoisan meaning ‘place of plenty’, -ensis from Greek meaning from.Holotype: AM 7999a (part), Fig. 10a.Figure 10(a–g) Mtshayelo kougaensis gen. et sp. nov., (h–i) Yarravia oblonga, (j–k) incertae sedis bilobed sporangia. (a) AM 7999a. Scale = 1 cm. Holotype. Bifurcating naked axis terminated by two synangiate structures. (b) Detail of AM 7999a. scale = 5 mm. Two synangiate structures. (c) Detail of AM 7999a. Scale = 5 mm. Detail showing the organization of a synangiate structure with the dehiscence line clearly visible. (d) Am 7902. Scale = 5 mm. Isolated synangiate structure. (e) AM 7904. Scale = 2.5 mm. Isolated branching axis showing two dichotomies and two terminal synangiate structures. (f) Detail of AM 7904. Scale = 5 mm. Two terminal synagiate structure. The arrow indicates where individual sporangia are starting. (g) AM 7990. Scale = 1 cm. Specimen showing several superimposed plants. (h) AM 7983. Scale = 5 mm. Gross morphology of the plant. (i) Detail of AM 7983. Scale = 5 mm. Detail of the synangiate structure showing four closely adpressed individual sporangia with a pointed tip. (j) AM 7933. Scale = 1 cm. Gross morphology of the plant. (k) Detail of AM 7933. Scale = 5 mm. Sporangia.Full size imageParatypes: AM 7902, AM 7904, AM 7933, AM 7983, AM 7990.Repository: Albany Museum, Devonian Lab, Beaufort Street, Makhanda, Eastern Cape, South Africa.Type locality: Impofu Dam, Kouga Municipality, Eastern Cape, South Africa (Fig. 1).Horizon: Kareedouw Member, Baviaanskloof Formation, Nardouw Subgroup, Table Mountain Group, Cape Supergroup.Age: Lower Devonian, Lochkovian?
Fig. 10a–g, Fig. 11.

Figure 11Proposed reconstruction of Mtshaelo kougaensis (a) and of its synangiate structure (b).Full size image
MaterialFive specimens of this plant have been collected from the LPL.DescriptionThis plant most often occurs as isolated branched axes (Fig. 10a–f) but can in some cases occur as densely packed trusses of axes (Fig. 10g). In the latter the organization of the branching system is obscured by the many superimpositions. The plant consists of robust smooth parallel-sided axes that dichotomize up to at least two times and terminate in trusses of elongate structures. The lack of preserved anatomy or spore contents prevents demonstration of the fertile nature of these structures. Considering their position and in analogy with other plants of similar organization we will consider them as sporangia. All sporangia seem to be attached at the same level giving to the whole structure the aspect of a synangium.The organization of the vegetative parts is best seen in specimen AM 7999 (Fig. 10a). This specimen is 80 mm long and has a generally flexuous appearance despite the robust aspect of the axes. It branches at least two times but only two synangium-like structures are preserved, only one of which exhibits clear attachment to the full branching system. The first order axis is 17 mm long and 1.4 mm wide. It branches at an angle of 32°. Second order axes are both quite flexuous and marked by a 90° curvature in the same direction. The left hand more completely preserved axis measures 20 mm long and 1.4 mm wide. It is difficult to say whether the evident curvature was originally present or the consequence of taphonomical processes. It could represent a horizontal part of the sporophyte as proposed in Fig. 4.Only one third order axis is fully exposed. It measures 39 mm long and is1.2 mm wide. Its width remains constant up to the distal end where it flares slightly up to 2.2. A second less complete termination flares to 2.6 mm from a subtending axis 1.0 mm wide. The axes then give the impression of being subdivided into several elongated structures that we interpret as synangiate sporangia.Several terminal structures are preserved (Fig. 10a–g). The distal end of the ultimate axis is marked by a slight and progressive widening (Fig. 10b–e). The axis then gives rise to several elongate sporangia that are all attached at the same level (Fig. 10d,e). The detailed organization of the termination is difficult to decipher. Although truncated, individual sporangia are particularly visible on the lower specimen in Fig. 10b. In this case three sporangia are visible and separated by a darker line of sediment. In other specimens, up to 4 sporangia can be identified (Fig. 10d,e). Distally, several additional tips can be seen suggesting that there are more sporangia hidden behind. Three to four additional tips can be identified in some cases (Fig. 10d,e). We consequently interpret the termination as being of 6 to 8 sporangia very likely organized in a circle. Individual sporangia are 0.4 to 0.8 mm wide and 4–6 mm long. They are spindle-shaped in profile, truncated proximally at the site of attachment and tapering distally to an acute tip. In some cases a slightly darker line can be observed within the sporangia. It could be interpreted as a longitudinal dehiscence line (Fig. 10c).
Yarravia64
Yarravia oblonga64
Fig. 10h and i
MaterialOne specimen from the LPL.DescriptionThis plant consists of robust smooth parallel-sided axes (Fig. 10h). Only one dichotomy is preserved. The axes are terminated by a truss of sporangia resembling a synangium. This specimen is 23 mm long and only shows the ultimate dichotomy and the axes subtending the terminal structures. The penultimate axis order is 9.8 mm long and 0.8 mm wide. It dichotomizes at an angle of 58° forming two axes measuring 4.5 and 7.0 mm long and 1.0 and 1.1 mm wide respectively. Only one synangium-like structure is preserved. Just before the insertion point of the sporangia, the subtending axis widens to 2.1 mm. Three elongate sporangia appear to all be inserted at the same level and are 3.5–4.2 mm long and 0.7–1.0 mm wide (Fig. 10i). They are parallel-sided. Their apices narrow abruptly and terminate in a slightly recurved beak-like structure. The tip of a possible fourth distal sporangium is also apparent.Identity and comparisonPlants presenting dichotomizing axes terminated by synangiate structures are rare in the Lower Devonian. The Impofu Dam material strongly recalls material attributed to the genus Yarravia that was reported from several Lower Devonian localities in Australia65,28,57 . Two species were identified Yarravia oblonga and Yarravia subsphaerica. The Impofu Dam material conforms more strongly to Yarravia oblonga. Of this plant, only the synangium-like structures and part of their subtending axes are known. The subtending axes present a massive aspect like that observed in the Impofu Dam material and the synangium like structures share the same organization. The size of the South African specimen is however smaller than the original material from Yarra Track but conforms almost exactly to material referred to as Yarravia cf. oblonga from Lilydale. Other occurrences of the genus Yarravia have been reported from France and Russia66,67, however these specimens need further study in order to be properly compared. Finally, specimens attributed to the genus Yarravia have been collected from the Devonian of Arizona but would as well need additional investigation68.
Incertae sedis heart-shape termination.
Fig. 10j and k.
MaterialA single specimen of this plant has been recovered from LPL.DescriptionThe plant measures 47 mm long. The branching system is apparently isotomous and branches only once (Fig. 10j). The first order is 33 mm long and 0.6 mm wide, slender and smooth. Second order are 9.2 and 9.8 mm long and 0.5 mm wide. The apex of each ultimate axis consists of a heart-shape structure measuring 4.0 mm long and 2.3 to 2.6 mm in width (Fig. 10k). This structure is complex and comprised of at least two more or less independent units. The two structures are easy to distinguish however they never seem to separate completely before their tips. These structures, here interpreted as sporangia, seem to occur after a dichotomy of the axis. Each one then progressively widens to reach 1.0 to 1.3 mm wide at two third of its length. Thereafter it forms a rounded tip, giving to the whole sporangium a club-shape.Identity and comparisonsThis plant bears a superficial resemblance to the Australian Yarravia Lang and Cookson28. Yarravia is characterised by several elongated sporangia apparently all attached at a single point. Our material rather seems to be composed of only two sporangia. A similar organisation was also described from the Lochkovian north Brown Clee Hill locality, Welsh Borderland (UK)60,61,62 . Although much smaller and preserved anatomically, Grisellatheca salopensis Edwards et al.62 presents a heart-shape fertile region made of two sporangia. Further comparison is however made difficult by the lack of anatomical details in our material. More

Mesocosm setupThe mesocosm experiment AQUACOSM VIMS-Ehux was carried out between 24th May (day 0) and 16th June (day 23) 2018 in Raunefjorden at the Marine Biological Station Espegrend, Norway (60°16′11 N; 5°13′07E) as previously described [7]. Four light-transparent enclosure bags were filled with surrounding fjord water (day −1; pumped from 5 m depth), and continuously mixed by aeration (from day 0 onwards). Each bag was supplemented with nutrients at a nitrogen to phosphorous ratio of 16:1 (1.6 µM NaNO3 and 0.1 µM KH2PO4 final concentration) on days 0–5 and 14–17, whereas on days 6, 7, and 13 only nitrogen was added. Nutrient concentrations were measured daily [18].Enumeration of phytoplankton cells by flow cytometryFor E. huxleyi enumeration by flow cytometry, water samples were collected in 50 mL tubes from ~1 m depth. Water samples were pre-filtered using 40 µm cell strainers and immediately analyzed with an Eclipse iCyt flow cytometer (Sony Biotechology, Champaign, IL, USA) as previously described [19]. A total volume of 300 µl with a flow rate of 150 µl min−1 was analyzed. A threshold was applied on the forward scatter to reduce background noise. Four groups of phytoplankton populations were identified in distinct gates by plotting the autofluorescence of chlorophyll (em: 663–737 nm) versus phycoerythrin (em: 570–620 nm) and side scatter: calcified E. huxleyi (high chlorophyll and high side scatter), Synechococcus (high phycoerythrin), nanophytoplankton including calcified and non-calcified E. huxleyi (high chlorophyll and phycoerythrin), and picophytoplankton (low chlorophyll and low phycoerythrin) [20]. See Fig. S1 for further details of gating strategy.Enumeration of EhV-like particles and bacteria by flow cytometryFor EhV and bacteria counts, 200 µl of sample were fixed a final concentration of 0.5% glutaraldehyde for one hour at 4 °C and flash frozen in liquid nitrogen. For analysis, they were thawed and stained with SYBR gold (Invitrogen, Carlsbad, CA, USA) that was diluted 1:10,000 in 0.2 μm filtered TE buffer (10:1 mM Tris:EDTA, pH 8), incubated for 20 min at 80 °C and cooled to room temperature [21]. Bacteria and EhV-like particles were counted and analyzed using an Eclipse iCyt flow cytometer (ex: 488 nm, em: 500–550 nm), and identified by comparing to reference samples containing fixed EhV201 and bacteria from lab cultures. EhV gating was very stringent in order to minimize the misidentification of other large viruses such as Micromonas pusilla virus (MpV) in the samples (see Fig. S2 for further details of gating strategy for EhV counts).Enumeration of extracellular EhV by qPCRWater samples (1–2 l) were sequentially filtered by vacuum through polycarbonate filters with a pore size of 20 µm (47 mm; Sterlitech, Kent, WA, US), then 2 µm (Isopore, 47 mm; Merck Millipore, Cork, Ireland), and finally 0.22 µm (Isopore, 47 mm; Merck Millipore). Filters were immediately flash-frozen in liquid nitrogen and stored at −80 °C until further processing. DNA was extracted from the 0.22 µm filters using the DNeasy PowerWater kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Each sample was diluted 100 times, and 1 µl was then used for qPCR analysis. EhV abundance was determined by qPCR for the major capsid protein (mcp) gene [22] using the following primers: 5ʹ-acgcaccctcaatgtatggaagg-3ʹ (mcp1F[23],) and 5ʹ-rtscrgccaactcagcagtcgt-3ʹ (mcp94Rv; Mayers, K. et al., unpublished). All reactions were carried out in technical triplicates. For all reactions, Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) was used as described by the manufacturer. Reactions were performed on a QuantStudio 5 Real-Time PCR System equipped with the QuantStudio Design and Analysis Software version 1.5.1 (Applied Biosystems, Foster City, CA, USA) as follows: 50 °C for 2 min, 95 °C for 5 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Results were calibrated against serial dilutions of EhV201 DNA at known concentrations, enabling exact enumeration of viruses. Samples showing multiple peaks in melting curve analysis or peaks that were not corresponding to the standard curves were omitted.Vesicle concentration and separationLab samplesE. huxleyi CCMP2090 was grown in 20 l filtered sea water (FSW) supplemented with K/2 nutrient mix at 18 °C, 16:8 h light:dark cycle, 100 μmol photons m−2 s−1. Uninfected cultures were grown to ~ 106 cells ml−1. Infected cultures were inoculated with EhV201 at a multiplicity of infection (MOI) of ~1:1 plaque forming unit (pfu) per cell and incubated under normal growth conditions for 120 h, at which time the culture had cleared. The entire 20 l volume was then filtered through a GF/C filter (Whatman, Maidstone, United Kingdom) followed by an 0.45 µm PVDF filter (Durapore, Merck Millipore) to eliminate cells and cellular debris.Mesocosm samplesOn days 2, 4, 5, 8, 12, 15, 18, and 23 we collected 25 l from bags 1–4 and combined them to produce one sample of 100 l for each sampling time. The samples were pre-filtered using a 200 µm nylon mesh, and then filtered through a GF/C filter (Whatman) followed by an 0.45 µm PVDF filter (Durapore, Merck Millipore) to eliminate cells and cellular debris.Particle concentrationParticles in the flow-through from the filtration stage were concentrated on a 100 kDa tangential flow filter (Spectrumlabs, Repligen, Waltham, Massachusetts, USA) to a final volume of ~500 ml. At this stage, mesocosm samples were stored in the dark at +4 oC and shipped back to the home lab. All samples were further concentrated to a final volume of 1–2 ml using 100 kDa Amicon-ultra filters (Merck Millipore).Vesicle separationVesicles were separated from other particles (including viruses) using an 18–35% OptiPrep gradient (MilliporeSigma, St. Louis, Missouri, USA). Gradients were centrifuged in an ultracentrifuge for 12 h at 200,000 × g. Fractions (0.5 ml) were collected from the top of the gradient and the fraction material was cleaned by washing three times and resuspended in 0.02 µm-filtered FSW using 100 kDa Amicon-ultra filters (Merck Millipore). Vesicles were detected in fractions with densities of 1.05–1.07 g ml–1 (fractions 3–5 from the top).Vesicle concentration in samples from lab cultures was measured by NTA using the NanoSight NS300 instrument (Malvern Instruments, Malvern, UK) equipped with a 488 nm laser module and NTA V3.2 software. Samples were diluted so that each field of view contained 20–100 particles. Three 60 s videos were recorded for each biological replicate, representing different fields of view. All the videos for a given experiment were processed using identical settings (screen gain of one and detection threshold of five).RNA extraction and sequencingIn order to eliminate RNA molecules that are not packed into vesicles, we subjected vesicle samples to RNase treatment prior to RNA extraction. Samples were incubated for 60 min at 37 oC with 10 pg µl−1 of RNase A (Bio Basic, Toronto, Canada). RNase activity was inactivated by adding 100 unites of Protector RNase Inhibitor (Roche, Basel, Switzerland). Total RNA (including RNA from ~18 nucleotides or more) was extracted using the miRNeasy kit according to the manufacturer’s instructions (Qiagen). Libraries were prepared using the TruSeq Small RNA Library kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. Each sample was indexed twice with the same index, one with polynucleotide kinase I treatment (according to manufacturer’s instructions, NEB, Ipswich, Massachusetts, USA) and one without. After 15 cycles of PCR amplification, libraries were cleaned with the QIAquick PCR Purification Kit according to the manufacturer’s instructions (Qiagen). Libraries were sequenced on the NextSeq platform (Illumina).sRNA bioinformatics analysisLow-quality read ends were trimmed and adaptors were removed using the cutadapt program [24], version 1.18. Reads shorter than 17 bp after the trimming were removed from further analyses. The remaining reads were mapped to an E. huxleyi integrated reference transcriptome shortly described in [6] using the RSEM software [25], version 1.3.1, with the default option of bowtie, version 1.1.2 [26]. Genes that had at least 5 reads in any of the samples were selected. For the heatmap (Fig. 1d), read counts were scaled to one million reads mapped to the E. huxleyi transcriptome and log2 transformed.Effect of vesicles on natural populations—experimental design and analysisOn days 14 and 20 of the mesocosm experiment (blue and red arrows in Fig. 1a, respectively), we combined equal volumes of water samples from bags 1–4 and filtered them through a 10 µm nylon mesh to eliminate zooplankton predators. We then supplemented the natural populations with f/50 nutrient mix and divided them into flasks, each containing 10 ml. In total, 30 flasks were treated with vesicles from uninfected lab cultures of E. huxleyi CCMP2090, at a ratio of ~500 vesicles cell−1 (calcified E. huxleyi determined by flow cytometry), and then all flasks were incubated in a growth chamber (15 °C, 16:8 h light:dark cycle, 100 μmol photons m−2 s−1). Once a day, samples were taken for flow cytometric quantification of live cells (see “Enumeration of phytoplankton cells by flow cytometry” above), or fixed for virus and bacteria counts (see “Enumeration of EhV-like particles and bacteria by flow cytometry” above). For statistical analysis, we used two-tailed t test with equal variance.Decay rate of EhV virions- experimental design and analysisTo determine the decay rate of infectivity of natural EhV virions, water was sampled from bag 4 on day 18, at a time point when viral infection was detected (green cross in Fig. 1a). This sample was filtered through a 0.45 PVDF filter (Durapore, Merck Millipore) to eliminate algal and most bacteria cells. EhV-like particles were counted by flow cytometry as described above and divided into nine tubes, each containing 1 ml. Triplicate samples were either treated with vesicles from EhV201-infected (VirusVesicles) or uninfected (controlVesicles) lab cultures (see above) at a ratio of ten vesicles per EhV-like particle, or not treated at all. All tubes were incubated in an on-land mesocosm facility that mimics the light and temperature conditions found at ~ 1 m depth within the fjord water. We used the most probable number (MPN) method [27] to determine the half-life of EhV within these samples. Briefly, a series of five-fold dilutions was prepared for each sample. Each dilution (10 μl) was then added, in eight technical replicates, to 100 μl of exponentially growing E. huxleyi CCMP374 cultures in multi-well plates and incubated under normal growth conditions for five days. This was repeated for four consecutive days for all samples. Clearance (infection) of the cells in the multi-wells was measured using an EnSpireTM 2300 Multilabel Reader (PerkinElmer, Turku, Finland) set to in vivo fluorescence (ex:460 nm, em:680 nm). MPN was calculated using the MPN calculation program, version 5 [28]. For the samples treated with controlvesicles, we could only obtain a positive MPN value for one time point, as the decay was faster than expected. Therefore, the minimum detectable infectivity values were used in order to calculate the maximum possible half-life. For statistical analysis, each treatment was compared to the untreated control, using ANOVA with Dunnett’s post-hoc test. More

1.Sokol, J. Troubled treasure. Fossils in Burmese amber offer an exquisite view of dinosaur times—and an ethical minefield. Science (23 May 2019).2.Lawton, G. Blood amber: the exquisite trove of fossils fueling war in Myanmar. New Scientist (1 May 2019).3.Lawton, G. Military now controls Myanmar’s scientifically important amber mines. New Scientist (30 August 2019).4.Rayfield, E. J., Theodor, J. M. & Polly, P. D. Fossils from conflict zones and reproducibility of fossil-based scientific data. Society of Vertebrate Paleontology (SVP) (21 April 2020).5.Barrett, P. M. & Johanson, Z. J. Syst. Palaeontol. 18, 1059 (2020).Article 

Google Scholar 
6.Engel, M. S. Nature 584, 525 (2020).CAS 
Article 

Google Scholar 
7.Barrett, P. M. & Johanson, Z. Nature 674, 586 (2020).
Google Scholar 
8.Poinar, G. & Ellenberger, S. Geoconserv. Res 3, 12–16 (2020).
Google Scholar 
9.Takai, M. et al. J. Hum. Evol. 84, 1–15 (2015).Article 

Google Scholar 
10.Barber, A. J., Khin Zaw & Crow, M. J. (eds) Myanmar: Geology, Resources and Tectonics Geological Society, London, Memoirs Vol. 48 (Geological Society of London, 2017).11.Jaeger, J.-J. et al. Proc. R. Soc. B 287, 20202129 (2020).Article 

Google Scholar 
12.Khin Zaw, Win Swe, Barber, A. J., Crow, M. J. & Yin Yin Nwe in Myanmar: Geology, Resources and Tectonics Geological Society, London, Memoirs Vol. 48 (eds Barber, A. J., Khin Zaw & Crow, M. J.) 1–17 (Geological Society of London, 2017).13.Phyo, M. M. et al. Minerals 10, 195 (2020).CAS 
Article 

Google Scholar 
14.GIAC (Geodynamics of India-Asia Collision) Final Report, A Joint Project of Scientific Co-operation between Total Myanmar Exploration and Production (TMEP), Unocal, Universities of Myanmar and Thailand, Myanmar Oil and Gas Enterprise (MOGE) and Ecole Normale Superieure (ENS) (TMEP, Unocal, Univ. Myanmar, Univ. Thailand, MOGE, ENS, 1999). More

College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao, ChinaChao Shi, Hao-hong Cai, Ri-xin Jiang & Shuo WangKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, ChinaChao Shi & Hua PengDepartment of Ecology & Evolutionary Biology, University of Kansas, Lawrence, KS, USAMichael S. EngelShanghai World Expo Museum, Shanghai, ChinaJi YuanKey Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaMing BaiDepartment of Entomology, College of Plant Protection, China Agricultural University, Beijing, ChinaDing YangCollege of Life and Environmental Sciences, Minzu University of China, Beijing, ChinaChun-lin LongCollege of Life Science, Shandong Normal University, Jinan, ChinaZun-tian ZhaoSouth China Botanical Garden, Chinese Academy of Sciences, Guangzhou, ChinaDian-xiang ZhangState Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, ChinaXian-chun ZhangState Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology and Center for Excellence in Life and Palaeoenvironment, Chinese Academy of Sciences, Nanjing, ChinaYong-dong WangSchool of Environment, Earth, and Ecosystem Sciences, The Open University, Milton Keynes, UKRobert A. SpicerCAS Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, ChinaRobert A. SpicerS.W., M.S.E., D.-X.Z., X.-C.Z., H.P., Y.-D.W. and R.A.S. conceived the idea and drafted the initial manuscript, with contributions from all other authors. All authors jointly revised the paper. More

1.Koch, E. & McFall-Ngai, M. Model systems for the study of how symbiotic associations between animals and extracellular bacterial partners are established and maintained. Drug Discov. Today Dis. Models 28, 3–12 (2018).PubMed 
Article 

Google Scholar 
2.Lee, K. H. & Ruby, E. G. Effect of the squid host on the abundance and distribution of symbiotic Vibrio fischeri in nature. Appl. Environ. Microbiol. 60, 1565–1571 (1994).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
3.Kremer, N. et al. Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization. Cell Host Microbe 14, 183–194 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
4.Bongrand, C. & Ruby, E. G. Achieving a multi-strain symbiosis: strain behavior and infection dynamics. ISME J. 13, 698–706 (2019).PubMed 
Article 

Google Scholar 
5.McFall-Ngai, M. J. The importance of microbes in animal development: lessons from the squid-vibrio symbiosis. Annu. Rev. Microbiol. 68, 177–194 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
6.Jones, B. W. & Nishiguchi, M. K. Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda). Mar. Biol. 144, 1151–1155 (2004).Article 

Google Scholar 
7.Graf, J. & Ruby, E. G. Host-derived amino acids support the proliferation of symbiotic bacteria. Proc. Natl Acad. Sci. USA 95, 1818–1822 (1998).CAS 
PubMed 
Article 

Google Scholar 
8.McFall-Ngai, M. J. & Ruby, E. G. Developmental biology in marine invertebrate symbioses. Curr. Opin. Microbiol. 3, 603–607 (2000).CAS 
PubMed 
Article 

Google Scholar 
9.Moriano-Gutierrez, S. et al. The noncoding small RNA SsrA is released by Vibrio fischeri and modulates critical host responses. PLoS Biol. 18, e3000934 (2020).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
10.Schwartzman, J. A. & Ruby, E. G. Stress as a normal cue in the symbiotic environment. Trends Microbiol. 24, 414–424 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
11.Nyholm, S. V. & McFall-Ngai, M. J. A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner. Nat. Rev. Microbiol. https://doi.org/10.1038/s41579-021-00567-y (2021).12.Schwartzman, J. A. et al. The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation. Proc. Natl Acad. Sci. USA 112, 566–571 (2015). In this study, the host’s delivery of chitin-derived N-acetylglucosamine is shown to develop 4 weeks after hatching, and this chitin is apparently delivered by haemocytes that lyse in the crypts only at night. A nocturnal acidification of the crypts results, and a model for how this outcome enhances bioluminescence is described.CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
13.Heath-Heckman, E. A. et al. Bacterial bioluminescence regulates expression of a host cryptochrome gene in the squid-vibrio symbiosis. mBio https://doi.org/10.1128/mBio.00167-13 (2013).Article 
PubMed 
PubMed Central 

Google Scholar 
14.Koropatnick, T. A. et al. Microbial factor-mediated development in a host-bacterial mutualism. Science 306, 1186–1187 (2004).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
15.Bouskra, D. et al. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–510 (2008).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
16.Thaiss, C. A. et al. Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis. Cell 159, 514–529 (2014).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
17.Ruby, E. G. Symbiotic conversations are revealed under genetic interrogation. Nat. Rev. Microbiol. 6, 752–762 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
18.Bongrand, C. & Ruby, E. G. The impact of Vibrio fischeri strain variation on host colonization. Curr. Opin. Microbiol. 50, 15–19 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
19.Colton, D. M. & Stabb, E. V. Rethinking the roles of CRP, cAMP, and sugar-mediated global regulation in the Vibrionaceae. Curr. Genet. 62, 39–45 (2016).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
20.Mandel, M. J. & Dunn, A. K. Impact and Influence of the natural Vibrio-squid symbiosis in understanding bacterial-animal interactions. Front. Microbiol. 7, 1982 (2016).PubMed 
PubMed Central 
Article 

Google Scholar 
21.Aschtgen, M. S. et al. Insights into flagellar function and mechanism from the squid-vibrio symbiosis. NPJ Biofilms Microbiomes 5, 32 (2019).PubMed 
PubMed Central 
Article 

Google Scholar 
22.Stabb, E. V. & Visick, K. L. in The Prokaryotes (eds Rosenberg, E., DeLong, E. F., Lory, S., Stackebrandt, E. & Thompson, F.) 497–532 (Springer, 2013).23.Nawroth, J. C. et al. Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome. Proc. Natl Acad. Sci. USA 114, 9510–9516 (2017). This work provides the first glimpse into the cilium-driven fluid mechanics that position V. fischeri cells to reach and settle in ‘quiet zones’ on the light organ surface, permitting a selective ‘recruitment’ of this microorganism from the planktonic environment.CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
24.Altura, M. A. et al. The first engagement of partners in the Euprymna scolopes-Vibrio fischeri symbiosis is a two-step process initiated by a few environmental symbiont cells. Environ. Microbiol. 15, 2937–2950 (2013). Aggregations of only a few V. fischeri cells are observed to initiate normal host responses, and reveal that aggregation is a two-part process that begins with bacterial attachment to the cilia.PubMed 
PubMed Central 

Google Scholar 
25.Nyholm, S. V., Stabb, E. V., Ruby, E. G. & McFall-Ngai, M. J. Establishment of an animal-bacterial association: recruiting symbiotic vibrios from the environment. Proc. Natl Acad. Sci. USA 97, 10231–10235 (2000).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
26.Yip, E. S., Geszvain, K., DeLoney-Marino, C. R. & Visick, K. L. The symbiosis regulator RscS controls the syp gene locus, biofilm formation and symbiotic aggregation by Vibrio fischeri. Mol. Microbiol. 62, 1586–1600 (2006).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
27.Koehler, S. et al. The model squid-vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement. Environ. Microbiol. https://doi.org/10.1111/1462-2920.14392 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
28.Morris, A. R. & Visick, K. L. Control of biofilm formation and colonization in Vibrio fischeri: a role for partner switching? Environ. Microbiol. 12, 2051–2059 (2010).CAS 
PubMed 
PubMed Central 

Google Scholar 
29.Norsworthy, A. N. & Visick, K. L. Gimme shelter: how Vibrio fischeri successfully navigates an animal’s multiple environments. Front. Microbiol. 4, 356 (2013).PubMed 
PubMed Central 
Article 

Google Scholar 
30.Shibata, S., Yip, E. S., Quirke, K. P., Ondrey, J. M. & Visick, K. L. Roles of the structural symbiosis polysaccharide (syp) genes in host colonization, biofilm formation, and polysaccharide biosynthesis in Vibrio fischeri. J. Bacteriol. 194, 6736–6747 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
31.Yip, E. S., Grublesky, B. T., Hussa, E. A. & Visick, K. L. A novel, conserved cluster of genes promotes symbiotic colonization and sigma-dependent biofilm formation by Vibrio fischeri. Mol. Microbiol. 57, 1485–1498 (2005).CAS 
PubMed 
Article 

Google Scholar 
32.Bassis, C. M. & Visick, K. L. The cyclic-di-GMP phosphodiesterase BinA negatively regulates cellulose-containing biofilms in Vibrio fischeri. J. Bacteriol. 192, 1269–1278 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
33.Chavez-Dozal, A., Hogan, D., Gorman, C., Quintanal-Villalonga, A. & Nishiguchi, M. K. Multiple Vibrio fischeri genes are involved in biofilm formation and host colonization. FEMS Microbiol. Ecol. 81, 562–573 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
34.Tischler, A. H., Lie, L., Thompson, C. M. & Visick, K. L. Discovery of calcium as a biofilm-promoting signal for Vibrio fischeri reveals new phenotypes and underlying regulatory complexity. J. Bacteriol. 200, e00016–e00018 (2018). This article expands our understanding of the regulatory controls and signals leading to biofilm formation by identifying calcium as a signal that induces a coordinate upregulation of Syp- and cellulose-dependent biofilm formation and revealing the sensor kinase HahK as a new biofilm regulator.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
35.Ziemba, C., Shabtai, Y., Piatkovsky, M. & Herzberg, M. Cellulose effects on morphology and elasticity of Vibrio fischeri biofilms. NPJ Biofilms Microbiomes 2, 1 (2016).PubMed 
PubMed Central 
Article 

Google Scholar 
36.Ray, V. A., Driks, A. & Visick, K. L. Identification of a novel matrix protein that promotes biofilm maturation in Vibrio fischeri. J. Bacteriol. 197, 518–528 (2015).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
37.Shibata, S. & Visick, K. L. Sensor kinase RscS induces the production of antigenically distinct outer membrane vesicles That depend on the symbiosis polysaccharide locus in Vibrio fischeri. J. Bacteriol. 194, 185–194 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
38.Hussa, E. A., Darnell, C. L. & Visick, K. L. RscS functions upstream of SypG to control the syp locus and biofilm formation in Vibrio fischeri. J. Bacteriol. 190, 4576–4583 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
39.Mandel, M. J., Wollenberg, M. S., Stabb, E. V., Visick, K. L. & Ruby, E. G. A single regulatory gene is sufficient to alter bacterial host range. Nature 458, 215–218 (2009).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
40.Ray, V. A., Eddy, J. L., Hussa, E. A., Misale, M. & Visick, K. L. The syp enhancer sequence plays a key role in transcriptional activation by the sigma54-dependent response regulator SypG and in biofilm formation and host colonization by Vibrio fischeri. J. Bacteriol. 195, 5402–5412 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
41.Visick, K. L. & Skoufos, L. M. Two-component sensor required for normal symbiotic colonization of Euprymna scolopes by Vibrio fischeri. J. Bacteriol. 183, 835–842 (2001).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
42.Norsworthy, A. N. & Visick, K. L. Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont. Mol. Microbiol. 96, 233–248 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
43.Thompson, C. M., Marsden, A. E., Tischler, A. H., Koo, J. & Visick, K. L. Vibrio fischeri biofilm formation prevented by a trio of regulators. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.01257-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
44.Brooks, J. F. II & Mandel, M. J. The histidine kinase BinK Is a negative regulator of biofilm formation and squid colonization. J. Bacteriol. 198, 2596–2607 (2016).PubMed 
PubMed Central 
Article 

Google Scholar 
45.Pankey, M. S. et al. Host-selected mutations converging on a global regulator drive an adaptive leap by bacteria to symbiosis. eLife https://doi.org/10.7554/eLife.24414 (2017). Evolutionary pathways that can lead to symbiotic colonization are revealed in this elegant study that follows the serial passage of a non-colonizing strain through many E. scolopes juveniles, resulting in altered, symbiosis-competent strains.Article 

Google Scholar 
46.Morris, A. R., Darnell, C. L. & Visick, K. L. Inactivation of a novel response regulator is necessary for biofilm formation and host colonization by Vibrio fischeri. Mol. Microbiol. 82, 114–130 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
47.Morris, A. R. & Visick, K. L. The response regulator SypE controls biofilm formation and colonization through phosphorylation of the syp-encoded regulator SypA in Vibrio fischeri. Mol. Microbiol. 87, 509–525 (2013).CAS 
PubMed 
Article 

Google Scholar 
48.Brooks, J. F. II et al. Global discovery of colonization determinants in the squid symbiont Vibrio fischeri. Proc. Natl Acad. Sci. USA 111, 17284–17289 (2014). This large-scale investigation of colonization factors provides important information on genetic requirements for symbiosis and provides a wealth of data for hypothesis generation that will foster many subsequent studies.CAS 
PubMed 
Article 

Google Scholar 
49.Thompson, C. M. & Visick, K. L. Assessing the function of STAS domain protein SypA in Vibrio fischeri using a comparative analysis. Front. Microbiol. 6, 760 (2015).PubMed 
PubMed Central 
Article 

Google Scholar 
50.Rotman, E. R. et al. Natural strain variation reveals diverse biofilm regulation in squid-colonizing Vibrio fischeri. J. Bacteriol. https://doi.org/10.1128/JB.00033-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
51.Bongrand, C. et al. A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior. ISME J. 10, 2907–2917 (2016). This study of the genomes and behaviours of a collection of a number of squid symbionts propelled the field from the near-exclusive study of a single isolate, ES114, into new and exciting directions with the genomic sequencing of dominant strains that contain numerous additional genetic sequences and factors.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
52.Newell, P. D., Boyd, C. D., Sondermann, H. & O’Toole, G. A. A c-di-GMP effector system controls cell adhesion by inside-out signaling and surface protein cleavage. PLoS Biol. 9, e1000587 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
53.Christensen, D. G., Marsden, A. E., Hodge-Hanson, K., Essock-Burns, T. & Visick, K. L. LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV. Mol. Microbiol. 114, 742–761 (2020). This article addresses a major long-standing question concerning the initiation of the light organ association; specifically, how do aggregated V. fischeri cells release themselves and migrate into host tissue? One factor may be an adhesin-cleaving protease, which is kept in check by a c-di-GMP-responsive protein, and can promote symbiont dispersal from biofilms.CAS 
PubMed 
Article 

Google Scholar 
54.Fidopiastis, P. M. et al. Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization. J. Bacteriol. 194, 3995–4002 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
55.Davidson, S. K., Koropatnick, T. A., Kossmehl, R., Sycuro, L. & McFall-Ngai, M. J. No means ‘yes’ in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association. Cellul. Microbiol. 6, 1139–1151 (2004).CAS 
Article 

Google Scholar 
56.Wang, Y. et al. Vibrio fischeri flavohaemoglobin protects against nitric oxide during initiation of the squid-Vibrio symbiosis. Mol. Microbiol. 78, 903–915 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
57.Stabb, E. V. Should they stay or should they go? Nitric oxide and the clash of regulators governing Vibrio fischeri biofilm formation. Mol. Microbiol. 111, 1–5 (2019).CAS 
PubMed 

Google Scholar 
58.Thompson, C. M., Tischler, A. H., Tarnowski, D. A., Mandel, M. J. & Visick, K. L. Nitric oxide inhibits biofilm formation by Vibrio fischeri via the nitric oxide sensor HnoX. Mol. Microbiol. 111, 187–203 (2019). This publication provides insight into the complex role in symbiosis of the squid-produced defence molecule NO by uncovering its ability to inhibit biofilm formation via the NO sensor HnoX, a finding that suggests that NO may influence the location or timing of biofilm formation and/or promote dispersal during symbiotic initiation.CAS 
PubMed 
Article 

Google Scholar 
59.Singh, P., Brooks, J. F. II., Ray, V. A., Mandel, M. J. & Visick, K. L. CysK plays a role in Biofilm formation and colonization by Vibrio fischeri. Appl. Environ. Microbiol. 81, 5223–5234 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
60.Raina, J. B., Fernandez, V., Lambert, B., Stocker, R. & Seymour, J. R. The role of microbial motility and chemotaxis in symbiosis. Nat. Rev. Microbiol. 17, 284–294 (2019).CAS 
PubMed 
Article 

Google Scholar 
61.Brennan, C. A., DeLoney-Marino, C. R. & Mandel, M. J. Chemoreceptor VfcA mediates amino acid chemotaxis in Vibrio fischeri. Appl. Environ. Microbiol. 79, 1889–1896 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
62.Graf, J., Dunlap, P. V. & Ruby, E. G. Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri. J. Bacteriol. 176, 6986–6991 (1994).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
63.Millikan, D. S. & Ruby, E. G. FlrA, a sigma54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization. J. Bacteriol. 185, 3547–3557 (2003).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
64.Millikan, D. S. & Ruby, E. G. Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization. J. Bacteriol. 186, 4315–4325 (2004).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
65.Wolfe, A. J., Millikan, D. S., Campbell, J. M. & Visick, K. L. Vibrio fischeri sigma54 controls motility, biofilm formation, luminescence, and colonization. Appl. Environ. Microbiol. 70, 2520–2524 (2004).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
66.O’Shea, T. M. et al. Magnesium promotes flagellation of Vibrio fischeri. J. Bacteriol. 187, 2058–2065 (2005).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
67.Ruby, E. G. & Asato, L. M. Growth and flagellation of Vibrio fischeri during initiation of the sepiolid squid light organ symbiosis. Arch. Microbiol. 159, 160–167 (1993).CAS 
PubMed 
Article 

Google Scholar 
68.Beeby, M. et al. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold. Proc. Natl Acad. Sci. USA 113, E1917–E1926 (2016).CAS 
PubMed 
Article 

Google Scholar 
69.Deloney-Marino, C. R. & Visick, K. L. Role for cheR of Vibrio fischeri in the Vibrio-squid symbiosis. Can. J. Microbiol. 58, 29–38 (2012).CAS 
PubMed 
Article 

Google Scholar 
70.Ruby, E. G. et al. Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners. Proc. Natl Acad. Sci. USA 102, 3004–3009 (2005).CAS 
PubMed 
Article 

Google Scholar 
71.Nikolakakis, K., Monfils, K., Moriano-Gutierrez, S., Brennan, C. A. & Ruby, E. G. Characterization of the Vibrio fischeri fatty acid chemoreceptors, VfcB and VfcB2. Appl. Environ. Microbiol. 82, 696–704 (2015).PubMed 
Article 
CAS 

Google Scholar 
72.Mandel, M. J. et al. Squid-derived chitin oligosaccharides are a chemotactic signal during colonization by Vibrio fischeri. Appl. Environ. Microbiol. 78, 4620–4626 (2012). While it was long-expected that V. fischeri might sense and be attracted to squid-produced molecules to facilitate directed migration into the light organ crypts, this work is the first to identify squid-produced molecules, chitin oligosaccharides, that function as a chemotactic signal promoting colonization.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
73.Bennett, B. D., Essock-Burns, T. & Ruby, E. G. HbtR, a heterofunctional homolog of the virulence regulator TcpP, facilitates the transition between symbiotic and planktonic lifestyles in Vibrio fischeri. mBio https://doi.org/10.1128/mBio.01624-20 (2020). Comparisons of V. fischeri with the related pathogen Vibrio cholerae reveal that a regulator conserved among Vibrio spp. plays very different roles in the interactions of these two microorganisms with their respective hosts.Article 
PubMed 
PubMed Central 

Google Scholar 
74.Brennan, C. A. et al. A model symbiosis reveals a role for sheathed-flagellum rotation in the release of immunogenic lipopolysaccharide. eLife 3, e01579 (2014). A surprising role for flagellar rotation in the release of lipopolysaccharide molecules that promote squid development is revealed in this work, providing a novel function for the flagellar sheath.PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
75.Stabb, E. V. & Millikan, D. S. in Defensive Mutualism in Microbial Symbiosis Vol. 27 (eds White, J. F. & Torres, M. S.) 85–98 (CRC Press, 2009).76.Bose, J. L., Rosenberg, C. S. & Stabb, E. V. Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture. Arch. Microbiol. 190, 169–183 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
77.Visick, K. L., Foster, J., Doino, J., McFall-Ngai, M. & Ruby, E. G. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J. Bacteriol. 182, 4578–4586 (2000).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
78.Verma, S. C. & Miyashiro, T. Niche-specific impact of a symbiotic function on the persistence of microbial symbionts within a natural host. Appl. Environ. Microbiol. 82, 5990–5996 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
79.Dunn, A. K., Millikan, D. S., Adin, D. M., Bose, J. L. & Stabb, E. V. New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl. Environ. Microbiol. 72, 802–810 (2006).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
80.Essock-Burns, T., Bongrand, C., Goldman, W. E., Ruby, E. G. & McFall-Ngai, M. J. Interactions of symbiotic partners drive the development of a complex biogeography in the squid-vibrio symbiosis. mBio 11, e00853-20 (2020).PubMed 
PubMed Central 
Article 

Google Scholar 
81.Sycuro, L. K., Ruby, E. G. & McFall-Ngai, M. Confocal microscopy of the light organ crypts in juvenile Euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis. J. Morphol. 267, 555–568 (2006).PubMed 
Article 
PubMed Central 

Google Scholar 
82.Koch, E. J., Miyashiro, T., McFall-Ngai, M. J. & Ruby, E. G. Features governing symbiont persistence in the squid-vibrio association. Mol. Ecol. 23, 1624–1634 (2014).PubMed 
Article 
PubMed Central 

Google Scholar 
83.Wollenberg, M. S., Preheim, S. P., Polz, M. F. & Ruby, E. G. Polyphyly of non-bioluminescent Vibrio fischeri sharing a lux-locus deletion. Environ. Microbiol. 14, 655–668 (2012).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
84.Chun, C. K. et al. Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association. Proc. Natl Acad. Sci. USA 105, 11323–11328 (2008).CAS 
PubMed 
Article 

Google Scholar 
85.McFall-Ngai, M., Heath-Heckman, E. A., Gillette, A. A., Peyer, S. M. & Harvie, E. A. The secret languages of coevolved symbioses: insights from the Euprymna scolopes-Vibrio fischeri symbiosis. Semin. Immunol. 24, 3–8 (2012).PubMed 
Article 

Google Scholar 
86.Moriano-Gutierrez, S. et al. Critical symbiont signals drive both local and systemic changes in diel and developmental host gene expression. Proc. Natl Acad. Sci. USA 116, 7990–7999 (2019).CAS 
PubMed 
Article 

Google Scholar 
87.Verma, S. C. & Miyashiro, T. Quorum sensing in the squid-Vibrio symbiosis. Int. J. Mol. Sci. 14, 16386–16401 (2013).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
88.Stabb, E. V., Schaefer, A., Bose, J. L. & Ruby, E. G. in Chemical Communication Among Bacteria (eds Winans, S. C. & Bassler, B. L.) 233–250 (ASM Press, 2008).89.Lupp, C. & Ruby, E. G. Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors. J. Bacteriol. 187, 3620–3629 (2005).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
90.Kimbrough, J. H. & Stabb, E. V. Comparative analysis reveals regulatory motifs at the ainS/ainR pheromone-signaling locus of Vibrio fischeri. Sci. Rep. 7, 11734 (2017).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
91.Kimbrough, J. H. & Stabb, E. V. Substrate specificity and function of the pheromone receptor AinR in Vibrio fischeri ES114. J. Bacteriol. 195, 5223–5232 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
92.Studer, S. V., Mandel, M. J. & Ruby, E. G. AinS quorum sensing regulates the Vibrio fischeri acetate switch. J. Bacteriol. 190, 5915–5923 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
93.Cao, X. et al. The novel sigma factor-like regulator RpoQ controls luminescence, chitinase activity, and motility in Vibrio fischeri. mBio https://doi.org/10.1128/mBio.00285-11 (2012).Article 
PubMed 
PubMed Central 

Google Scholar 
94.Studer, S. V. et al. Non-native acylated homoserine lactones reveal that LuxIR quorum sensing promotes symbiont stability. Environ. Microbiol. 16, 2623–2634 (2014).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
95.Boettcher, K. J. & Ruby, E. G. Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes. J. Bacteriol. 172, 3701–3706 (1990).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
96.Septer, A. N. & Stabb, E. V. Coordination of the arc regulatory system and pheromone-mediated positive feedback in controlling the Vibrio fischeri lux operon. PLoS ONE 7, e49590 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
97.Stabb, E. V. Could positive feedback enable bacterial pheromone signaling to coordinate behaviors in response to heterogeneous environmental cues? mBio https://doi.org/10.1128/mBio.00098-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
98.Bose, J. L. et al. Bioluminescence in Vibrio fischeri is controlled by the redox-responsive regulator ArcA. Mol. Microbiol. 65, 538–553 (2007).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
99.Lyell, N. L. et al. Cyclic AMP receptor protein regulates pheromone-mediated bioluminescence at multiple levels in Vibrio fischeri ES114. J. Bacteriol. 195, 5051–5063 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
100.Lyell, N. L., Dunn, A. K., Bose, J. L. & Stabb, E. V. Bright mutants of Vibrio fischeri ES114 reveal conditions and regulators that control bioluminescence and expression of the lux operon. J. Bacteriol. 192, 5103–5114 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
101.Septer, A. N., Lyell, N. L. & Stabb, E. V. The iron-dependent regulator fur controls pheromone signaling systems and luminescence in the squid symbiont Vibrio fischeri ES114. Appl. Environ. Microbiol. 79, 1826–1834 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
102.Stoudenmire, J. L. et al. An iterative, synthetic approach to engineer a high-performance PhoB-specific reporter. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.00603-18 (2018). This study not only provides a road map for synthetic promoter engineering in V. fischeri but also uncovers evidence for possible microenvironments present within different crypts of the E. scolopes light organ.Article 
PubMed 
PubMed Central 

Google Scholar 
103.Bose, J. L. et al. Contribution of rapid evolution of the luxR-luxI intergenic region to the diverse bioluminescence outputs of Vibrio fischeri strains isolated from different environments. Appl. Environ. Microbiol. 77, 2445–2457 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
104.Dunn, A. K. Vibrio fischeri metabolism: symbiosis and beyond. Adv. Microb. Physiol. 61, 37–68 (2012).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
105.Schwartzman, J. A. & Ruby, E. G. A conserved chemical dialog of mutualism: lessons from squid and vibrio. Microbes Infect. 18, 1–10 (2016).PubMed 
Article 
PubMed Central 

Google Scholar 
106.Pan, S. et al. Model-enabled gene search (MEGS) allows fast and direct discovery of enzymatic and transport gene functions in the marine bacterium Vibrio fischeri. J. Biol. Chem. 292, 10250–10261 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
107.Thompson, L. R. et al. Transcriptional characterization of Vibrio fischeri during colonization of juvenile Euprymna scolopes. Environ. Microbiol. 19, 1845–1856 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
108.Wier, A. M. et al. Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis. Proc. Natl Acad. Sci. USA 107, 2259–2264 (2010). In the first dual transcriptional study of an animal host and its symbionts, gene expression in both partners is shown to be regulated over a day–night cycle, revealing a daily remodelling of the crypt epithelial cells and a night-time provision of chitin to the symbionts.CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
109.Sun, Y., Verma, S. C., Bogale, H. & Miyashiro, T. NagC represses N-acetyl-glucosamine utilization genes in Vibrio fischeri within the light organ of Euprymna scolopes. Front. Microbiol. 6, 741 (2015).PubMed 
PubMed Central 

Google Scholar 
110.Wasilko, N. P. et al. Sulfur availability for Vibrio fischeri growth during symbiosis establishment depends on biogeography within the squid light organ. Mol. Microbiol. 111, 621–636 (2019). This study sheds light on both the nutritional adaptability of V. fischeri and the complex biogeography of the light organ by demonstrating that this symbiont uses different sulfur sources within different regions of the light organ.CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
111.Septer, A. N. et al. Bright luminescence of Vibrio fischeri aconitase mutants reveals a connection between citrate and the Gac/Csr regulatory system. Mol. Microbiol. 95, 283–296 (2015).CAS 
PubMed 
Article 

Google Scholar 
112.Lyell, N. L. & Stabb, E. V. Symbiotic characterization of Vibrio fischeri ES114 mutants that display enhanced luminescence in culture. Appl. Environ. Microbiol. 79, 2480–2483 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
113.Lyell, N. L. et al. An expanded transposon mutant library reveals that Vibrio fischeri delta-aminolevulinate auxotrophs can colonize Euprymna scolopes. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.02470-16 (2017).Article 
PubMed 
PubMed Central 

Google Scholar 
114.Colton, D. M., Stoudenmire, J. L. & Stabb, E. V. Growth on glucose decreases cAMP-CRP activity while paradoxically increasing intracellular cAMP in the light-organ symbiont Vibrio fischeri. Mol. Microbiol. 97, 1114–1127 (2015).CAS 
PubMed 
Article 

Google Scholar 
115.Miyashiro, T. et al. The N-acetyl-D-glucosamine repressor NagC of Vibrio fischeri facilitates colonization of Euprymna scolopes. Mol. Microbiol. 82, 894–903 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
116.Adin, D. M., Visick, K. L. & Stabb, E. V. Identification of a cellobiose utilization gene cluster with cryptic beta-galactosidase activity in Vibrio fischeri. Appl. Environ. Microbiol. 74, 4059–4069 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
117.Pan, M., Schwartzman, J. A., Dunn, A. K., Lu, Z. & Ruby, E. G. A single host-derived glycan impacts key regulatory nodes of symbiont metabolism in a coevolved mutualism. mBio 6, e00811 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
118.Boettcher, K. J., McFall-Ngai, M. J. & Ruby, E. G. Bioluminescence in the symbiotic squid Euprymna scolopes is controlled by a daily biological rhythm. J. Comp. Physiol. 179, 65–73 (1996).Article 

Google Scholar 
119.Kremer, N. et al. The dual nature of haemocyanin in the establishment and persistence of the squid-vibrio symbiosis. Proc. Biol. Sci. 281, 20140504 (2014).PubMed 
PubMed Central 

Google Scholar 
120.Stabb, E. V. Shedding light on the bioluminescence “paradox”. ASM News 71, 223–229 (2005).
Google Scholar 
121.Septer, A. N., Bose, J. L., Dunn, A. K. & Stabb, E. V. FNR-mediated regulation of bioluminescence and anaerobic respiration in the light-organ symbiont Vibrio fischeri. FEMS Microbiol. Lett. 306, 72–81 (2010).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
122.Dunn, A. K. Alternative oxidase activity reduces stress in Vibrio fischeri cells exposed to nitric oxide. J. Bacteriol. https://doi.org/10.1128/JB.00797-17 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
123.Dunn, A. K. & Stabb, E. V. Genetic analysis of trimethylamine N-oxide reductases in the light organ symbiont Vibrio fischeri ES114. J. Bacteriol. 190, 5814–5823 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
124.Septer, A. N., Wang, Y., Ruby, E. G., Stabb, E. V. & Dunn, A. K. The haem-uptake gene cluster in Vibrio fischeri is regulated by Fur and contributes to symbiotic colonization. Environ. Microbiol. 13, 2855–2864 (2011).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
125.Graf, J. & Ruby, E. G. Novel effects of a transposon insertion in the Vibrio fischeri glnD gene: defects in iron uptake and symbiotic persistence in addition to nitrogen utilization. Mol. Microbiol. 37, 168–179 (2000).CAS 
PubMed 
Article 

Google Scholar 
126.Eickhoff, M. J. & Bassler, B. L. Vibrio fischeri siderophore production drives competitive exclusion during dual-species growth. Mol. Microbiol. 114, 244–261 (2020).CAS 
PubMed 
Article 

Google Scholar 
127.Ferretti, P. et al. Mother-to-infant microbial transmission from different body sites shapes the developing infant gut microbiome. Cell Host Microbe 24, 133–145 e135 (2018).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
128.Zheng, H. et al. Division of labor in honey bee gut microbiota for plant polysaccharide digestion. Proc. Natl Acad. Sci. USA 116, 25909–25916 (2019).CAS 
PubMed 
Article 
PubMed Central 

Google Scholar 
129.Aschtgen, M. S. et al. Rotation of Vibrio fischeri flagella produces outer membrane vesicles that induce host development. J. Bacteriol. 198, 2156–2165 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
130.Aschtgen, M. S., Wetzel, K., Goldman, W., McFall-Ngai, M. & Ruby, E. Vibrio fischeri-derived outer membrane vesicles trigger host development. Cell. Microbiol. 18, 488–499 (2016).CAS 
PubMed 
Article 

Google Scholar 
131.Lynch, J. B. et al. Ambient pH alters the protein content of outer membrane vesicles, driving host development in a beneficial symbiosis. J. Bacteriol. https://doi.org/10.1128/JB.00319-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
132.Franzenburg, S. et al. Distinct antimicrobial peptide expression determines host species-specific bacterial associations. Proc. Natl Acad. Sci. USA 110, E3730–E3738 (2013).CAS 
PubMed 
Article 

Google Scholar 
133.Chen, F. et al. Bactericidal permeability-increasing proteins shape host-microbe interactions. mBio 8, e00040-17 (2017).PubMed 
PubMed Central 
Article 

Google Scholar 
134.Heath-Heckman, E. A. et al. Shaping the microenvironment: evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-Vibrio symbiosis. Environ. Microbiol. 16, 3669–3682 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
135.Wang, Y. et al. H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri. Proc. Natl Acad. Sci. USA 107, 8375–8380 (2010).CAS 
PubMed 
Article 

Google Scholar 
136.Schwartzman, J. A. et al. Acidic pH promotes lipopolysaccharide modification and alters colonization in a bacteria-animal mutualism. Mol. Microbiol. 112, 1326–1338 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
137.Kwong, W. K. & Moran, N. A. Gut microbial communities of social bees. Nat. Rev. Microbiol. 14, 374–384 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
138.Murfin, K. E. et al. Xenorhabdus bovienii strain diversity impacts coevolution and symbiotic maintenance with Steinernema spp. nematode hosts. mBio 6, e00076 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
139.Wollenberg, M. S. & Ruby, E. G. Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from two Oahu (Hawaii) populations. Appl. Environ. Microbiol. 75, 193–202 (2009). This is the first comparative genome-level study of light organ symbionts both between and within adult squid, suggesting that on average each crypt of an organ is colonized by one or two V. fischeri cells, potentially creating crypt-separated, clonal lineages within a polyclonal organ.CAS 
PubMed 
Article 

Google Scholar 
140.Tomich, M., Planet, P. J. & Figurski, D. H. The tad locus: postcards from the widespread colonization island. Nat. Rev. Microbiol. 5, 363–375 (2007).CAS 
PubMed 
Article 

Google Scholar 
141.Gyllborg, M. C., Sahl, J. W., Cronin, D. C. III., Rasko, D. A. & Mandel, M. J. Draft genome sequence of Vibrio fischeri SR5, a strain isolated from the light organ of the Mediterranean squid Sepiola robusta. J. Bacteriol. 194, 1639 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
142.Bongrand, C. et al. Using colonization assays and comparative genomics to discover symbiosis behaviors and factors in Vibrio fischeri. mBio https://doi.org/10.1128/mBio.03407-19 (2020).Article 
PubMed 
PubMed Central 

Google Scholar 
143.Coryell, R. L. et al. Phylogeographic patterns in the Philippine archipelago influence symbiont diversity in the bobtail squid-Vibrio mutualism. Ecol. Evol. 8, 7421–7435 (2018).PubMed 
PubMed Central 
Article 

Google Scholar 
144.Soto, W., Rivera, F. M. & Nishiguchi, M. K. Ecological diversification of Vibrio fischeri serially passaged for 500 generations in novel squid host Euprymna tasmanica. Microb. Ecol. 67, 700–721 (2014).PubMed 
PubMed Central 
Article 

Google Scholar 
145.Soto, W., Travisano, M., Tolleson, A. R. & Nishiguchi, M. K. Symbiont evolution during the free-living phase can improve host colonization. Microbiology 165, 174–187 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
146.Fidopiastis, P. M., von Boletzky, S. & Ruby, E. G. A new niche for Vibrio logei, the predominant light organ symbiont of squids in the genus Sepiola. J. Bacteriol. 180, 59–64 (1998).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
147.Dillon, M. M., Sung, W., Lynch, M. & Cooper, V. S. Periodic variation of mutation rates in bacterial genomes associated with replication timing. mBio https://doi.org/10.1128/mBio.01371-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
148.Dillon, M. M., Sung, W., Sebra, R., Lynch, M. & Cooper, V. S. Genome-wide biases in the rate and molecular spectrum of spontaneous mutations in Vibrio cholerae and Vibrio fischeri. Mol. Biol. Evol. 34, 93–109 (2017).CAS 
PubMed 
Article 

Google Scholar 
149.Wollenberg, M. S. & Ruby, E. G. Phylogeny and fitness of Vibrio fischeri from the light organs of Euprymna scolopes in two Oahu, Hawaii populations. ISME J. 6, 352–362 (2012).CAS 
PubMed 
Article 

Google Scholar 
150.Koch, E. J. et al. The cytokine MIF controls daily rhythms of symbiont nutrition in an animal-bacterial association. Proc. Natl Acad. Sci. USA 117, 27578–27586 (2020).CAS 
PubMed 
Article 

Google Scholar 
151.Sun, Y. et al. Intraspecific competition impacts Vibrio fischeri strain diversity during initial colonization of the squid light organ. Appl. Environ. Microbiol. 82, 3082–3091 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
152.Speare, L. et al. Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host. Proc. Natl Acad. Sci. USA 115, E8528–E8537 (2018). The finding that V. fischeri engages in biological ‘warfare’ to become the sole colonizer of a given crypt has provided new insight into the dynamics and processes controlling light organ population structure and strain competition in nature.CAS 
PubMed 
Article 

Google Scholar 
153.Speare, L., Smith, S., Salvato, F., Kleiner, M. & Septer, A. N. Environmental viscosity modulates interbacterial killing during habitat transition. mBio https://doi.org/10.1128/mBio.03060-19 (2020).Article 
PubMed 
PubMed Central 

Google Scholar 
154.Guckes, K. R. et al. Incompatibility of Vibrio fischeri strains during symbiosis establishment depends on two functionally redundant hcp genes. J. Bacteriol. https://doi.org/10.1128/JB.00221-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
155.Guckes, K. R., Cecere, A. G., Williams, A. L., McNeil, A. E. & Miyashiro, T. The bacterial enhancer binding protein VasH promotes expression of a Type VI secretion system in Vibrio fischeri during symbiosis. J. Bacteriol. https://doi.org/10.1128/JB.00777-19 (2020).Article 
PubMed 
PubMed Central 

Google Scholar 
156.Bultman, K. M., Cecere, A. G., Miyashiro, T., Septer, A. N. & Mandel, M. J. Draft genome sequences of type VI secretion system-encoding Vibrio fischeri strains FQ-A001 and ES401. Microbiol. Resour. Announc. https://doi.org/10.1128/MRA.00385-19 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
157.Doino, J. A. & McFall-Ngai, M. J. A transient exposure to symbiosis-competent bacteria induces light organ morphogenesis in the host squid. Biol. Bull. 189, 347–355 (1995).CAS 
PubMed 
Article 

Google Scholar 
158.Dunn, A. K., Martin, M. O. & Stabb, E. V. Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri. Plasmid 54, 114–134 (2005).CAS 
PubMed 
Article 

Google Scholar 
159.Lyell, N. L., Dunn, A. K., Bose, J. L., Vescovi, S. L. & Stabb, E. V. Effective mutagenesis of Vibrio fischeri by using hyperactive mini-Tn5 derivatives. Appl. Environ. Microbiol. 74, 7059–7063 (2008).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
160.Stoudenmire, J. L., Black, M., Fidopiastis, P. M. & Stabb, E. V. Mutagenesis of Vibrio fischeri and other marine bacteria using hyperactive mini-Tn5 derivatives. Methods Mol. Biol. 2016, 87–104 (2019).CAS 
PubMed 
Article 

Google Scholar 
161.Pollack-Berti, A., Wollenberg, M. S. & Ruby, E. G. Natural transformation of Vibrio fischeri requires tfoX and tfoY. Environ. Microbiol. 12, 2302–2311 (2010).PubMed 
PubMed Central 

Google Scholar 
162.Visick, K. L., Hodge-Hanson, K. M., Tischler, A. H., Bennett, A. K. & Mastrodomenico, V. Tools for rapid genetic engineering of Vibrio fischeri. Appl. Environ. Microbiol. https://doi.org/10.1128/AEM.00850-18 (2018).Article 
PubMed 
PubMed Central 

Google Scholar 
163.Burgos, H. L. et al. Multiplexed competition in a synthetic squid light organ microbiome using barcode-tagged gene deletions. mSystems 5, e00846-20 (2020).PubMed 
PubMed Central 
Article 

Google Scholar 
164.Brooks, J. F. II, Gyllborg, M. C., Kocher, A. A., Markey, L. E. & Mandel, M. J. TfoX-based genetic mapping identifies Vibrio fischeri strain-level differences and reveals a common lineage of laboratory strains. J. Bacteriol. 197, 1065–1074 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
165.Califano, G. et al. Draft genome sequence of Aliivibrio fischeri strain 5LC, a bacterium retrieved from gilthead seabream (Sparus aurata) larvae reared in aquaculture. Genome Announc. 3, e00593-15 (2015).PubMed 
PubMed Central 
Article 

Google Scholar 
166.Hehemann, J.-H. et al. Adaptive radiation by waves of gene transfer leads to fine-scale resource partitioning in marine microbes. Nat. Commun. 7, 12860 (2016).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
167.Nikolakakis, K., Lehnert, E., McFall-Ngai, M. J. & Ruby, E. G. Use of hybridization chain reaction-fluorescent in situ hybridization to track gene expression by both partners during initiation of symbiosis. Appl. Environ. Microbiol. 81, 4728–4735 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar  More