Philippa Kaur

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Two modes of oviparity, i.e. single and multiple oviparity, are currently recognized in chondrichthyans1,2,3,5,6,7,8,9,10,11,12. Single oviparity (Fig. 3a) is a mode where each oviduct in pregnant females contains one egg case, i.e. a pair of egg cases in a pregnant female. These egg cases are retained in the oviduct only for a short time and deposited immediately before the embryo has begun developing. The embryos are not recognizable at oviposition and become visible in a few weeks. Oviposition is repeatedly performed, and each mature female can deposit tens of egg cases over the course of a spawning season6 (“Short single” oviparity in Fig. 4). Multiple oviparity (Fig. 3b) is a mode where several egg cases accumulate in each oviduct and are retained for several months before oviposition, in which time embryos begin development in the oviduct and the egg cases are deposited later when the embryos grow large to a certain developmental stage (“Multiple” oviparity in Fig. 4).
Figure 3

Three modes of oviparity in catsharks, showing egg cases in oviduct. (a) Short single oviparity (Galeus sauteri from Taiwan, uncatalogued), (b) multiple oviparity (Halalelurus buergeri, 410 mm TL from Kagoshima, Japan, uncatalogued), (c) Sustained single oviparity (Cephaloscyllium sarawakensis), (c1) egg cases without developing embryo (NMMB-P30890, 80.0 mm ECL), (c2) egg cases with a developing embryo in each (NMMB-P 30888, 81.6 mm, 83.0 mm ECL). Top three photographs (a,b,c1) cover whole abdominal cavities, showing difference of relative sizes of egg cases in three modes of oviparity. Cephaloscyllium sarawakensis (c1) has huge egg cases occupying most of the abdominal cavity.

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Figure 4

Schematic diagram of five modes of reproduction in catsharks, showing differences of succession, duration and condition of egg cases/ embryos per one oviduct/uterus. Numerals show order of egg case produced in the oviduct/uterus.

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Cephaloscyllium sarawakensis does not fit the classic single oviparity, nor multiple oviparity. Pregnant females of this species always have a single egg case, never two or more, in each oviduct (Fig. 3c), and keep it until embryo attains a certain developmental stage (“Sustained single” oviparity in Fig. 4). These facts indicate that reproduction of C. sarawakensis represents a new mode of oviparity, which is herein termed “sustained single oviparity”. A 450 mm TL female of Cephaloscyllium silasi from the Indian Ocean had one egg case with a well-developed embryo in each oviduct13. Although they reported only one female specimen, this species possibly also displays the sustained single oviparity.
Various technical terms have been used for oviparity. Single oviparity has at least three alternative names, “extended” oviparity3,4,6,12,14, “external” oviparity5,15, and “simple” oviparity5. Multiple oviparity has been also termed “retained” oviparity3,4,5,6,12,14,16. These terms are used for the same reproductive mode, and this could lead to confusion and misunderstanding about the oviparity. Therefore, we herein summarized the terms of oviparity and proposed a new set of technical terms as follows: (1) “short single oviparity” for the single oviparity previously known; (2) “sustained single oviparity” for the new type of oviparity reported in this study; and (3) “multiple oviparity” instead of the former “retained” oviparity. The new definition of oviparity in the cartilaginous fishes was summarized, with additions of two modes of yolk-sac viviparity in catsharks (Table 1).
Table 1 New definitions of oviparity and yolk-sac viviparity in lecithotrophic (yolk-dependent) cartilaginous fishes.
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Typically in the catsharks displaying short single oviparity, the shell of egg case is tough and thick, with two pairs of long strong tendrils on its anterior and posterior ends (Fig. 5a‒c,e,f), although tendrils are very short or replaced by fine silky materials (Fig. 5d) or absent in some species. The posterior pair of tendrils is used to attach the egg case to the substrate on the sea bottom, to pull it out from the oviduct, and coil it around the substrate, together with anterior pair of tendrils. The egg case is firmly secured on the substrate until juvenile hatches. The tendrils in the multiple oviparous species (Fig. 5f) tend to be thinner and shorter than those of the short single oviparous species. Cephaloscyllium sarawakensis of the sustained single oviparity has a thick shell and long tendrils (Fig. 1b,c,d1). The two egg cases (Fig. 2a) collected from fishery landings can be inferred to have been deposited intentionally on the tube of a tubeworm Paradiopatra sp. based on the fact that the posterior tendrils are firmly twined around it.
Figure 5

Egg cases of catsharks. (a) Cephaloscyllium laticeps from Australia, (b) Cephaloscyllium umbratile from Japan, (c) Poroderma africanum from Ibaraki Prefectural Oarai Aquarium, Japan, (d) Galeus sauteri from Taiwan, (e) Haploblepharus fuscus from Ibaraki Prefectural Oarai Aquarium, Japan, (f) Halaelurus buergeri from Japan. (a‒e) short single oviparous species, (f) multiple oviparous species. Scales 30 mm.

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The egg cases in the species of sustained single oviparity are very large, with its length (ECL) ranging 16.5‒20.1% TL in Cephaloscyllium sarawakensis and 18.9‒19.2% TL in C. silasi13, while the egg cases of short single oviparous C. umbratile are 10.6‒15.1% TL2,17,18. The egg cases of other short single oviparous species are also small, with lengths 8.6‒9.0% TL in Holohalaelurus regani19, 9.2% TL in Galeus sauteripresent study, 9.2‒14.8% TL in four species of Apristurus20,21,22,23, 10.6‒14.9% TL in Atelomycterus marmoratus24, 11.4% TL in Schroederichthys maculatus25, 11.5‒11.9% TL in Scyliorhinus torazame26,presentstudy, 11.8% TL in S. capensis27, 12.0% TL in Bythaelurus dawsoni28 and 13.5‒18.9% TL from Fig. 10d29 of Parmaturus xaniurus. The egg case lengths of multiple oviparous species are about 11% TL in Halaelurus buergeripresent study and 10.4‒10.9% TL in H. quagga30. As seen in Fig. 3, the egg cases of C. sarawakensis (Fig. 3c1) are far larger than those of G. sauteri (Fig. 3a) and H. buergeri (Fig. 3b), occupying most of the available space of oviduct and abdominal cavity. Thus, the two species of sustained single oviparity have much larger egg cases than short single oviparous and multiple oviparous species, suggesting larger neonates at hatching in C. sarawakensis and C. silasi.
The embryos in the egg cases recorded from the oviduct indicate that Cephaloscyllium sarawakensis retains the egg case until the embryo attains about 102 mm TL (Fig. 1d; largest embryo in the egg inside the oviduct), and then oviposition occurs later. However, the two egg cases (both 80.0 mm ECL) from the seabed contained a 65 mm TL and a 94.5 mm TL embryo each (Fig. 2a). These facts suggest the timing of oviposition is rather wide in this species, and the egg cases are laid when the embryo grows roughly 6‒10 + cm TL, stimulated by some internal or external factors. The smallest free-swimming juvenile collected was 125 mm TL with a remnant of external yolk-sac (Fig. 2b), suggesting the hatching size from egg case being around 120 mm TL in this species. These evidences indicate C. sarawakensis keeps the egg case in the oviduct until embryo has developed to 50 ~ 80+ % of its hatching size. Therefore, the retention of egg case in the oviduct continues for an extended period, perhaps several months or more, in C. sarawakensis and probably also in C. silasi.
The other remarkable characteristic of Cephaloscyllium sarawakensis is the glassy transparent egg cases (Fig. 1b‒d), and the transparency is completely maintained even after oviposition (Fig. 2a). The egg cases of oviparous cartilaginous fishes are opaque, usually yellowish to dark brownish (Fig. 5), and sometimes with longitudinal or transverse ridges (Fig. 5a). The functional role of egg case is to protect the embryo from physical, physiological and biological hazards from the environment. The colored egg cases can be also effective to conceal the embryo in it. However, the egg case of C. sarawakensis is transparent and never cryptic that the orange yellow yolk would clearly be recognizable through transparent egg case, if the egg case is deposited immediately after egg case being formed.
As shown in this study, C. sarawakensis retains an egg case in each oviduct until the embryo is developed with a distinct dark polka-dot color pattern on light brownish body (Figs. 1d, 2a), typically seen in the juveniles (Fig. 2b). One of the reasons for transparent egg case may be related to their vivid body color patterns. Benthic and reef-dwelling sharks have complicated color patterns, which are effective to blend the body into their background or for camouflage31. The present egg cases of C. sarawakensis (Fig. 2a) were deposited around the tube of a tubeworm sticking out from the seabed. Their vivid polka-dots and light brownish body coloration could function as more effective camouflage against the complex and dark background through the transparent egg case. Thus, the long retention of transparent egg cases and vivid embryonic coloration could suggest a new method of reproductive tactics in cartilaginous fishes.
The oviparity is advantageous as a method to increase the fecundity in small elasmobranchs that have limited space in body cavity for care and storage of the embryos6. The species of short single oviparity (Fig. 3a) repeatedly deposits two egg cases immediately after the cases are completed and laid, resulting in 20‒100 eggs per season6. The captive Cephaloscyllium umbratile was recorded as depositing two egg cases at intervals of 11‒38 days (20 days in average) for whole year32, which means a single female deposited about 36 egg cases a year. Similarly, the captive C. laticeps laid two egg cases at intervals of up to 28 days throughout whole year11, equating more than 26 egg cases being deposited annually.
The multiple oviparous species retains a number of egg cases in each oviduct (Fig. 3b) for several months until the embryos have developed to a certain stage. All the species of Halaelurus are multiple oviparous, and H. buergeri has been recorded to deposit 8 egg cases one by one at a stage when the embryos inside have attained 70 mm TL33, or 10 egg cases at one time34. One specimen of H. buergeri we (KN) collected (Fig. 3b) had 10 egg cases in the oviducts with a developing embryo in each. A captive H. maculosus deposited 11 egg cases containing 50‒70 mm TL embryos in five days (personal communication with Mr. K. Tokunaga of Ibaraki Prefectural Oarai Aquarium). The fecundity of H. lineatus is up to 16 eggs at a time35.
The maternal environment offers the best protection to the developing embryos and can shorten the exposed period of time on the substrate. Therefore, the survival rate could be expected to be much higher in the sustained single and multiple oviparous species than the short single oviparous species. The species of the sustained single oviparity (Fig. 3c) and those of the multiple oviparity (Fig. 3b) are the same in that the egg cases have long maternal protection, but the number of the egg cases deposited at a time is considerably less in the sustained single oviparous species, i.e. 2 eggs vs. 4‒16 eggs per mother, respectively. Hence, the fecundity of Cephaloscyllium sarawakensis could be very low, 1/8–1/2 of the multiple oviparous species (see “Oviparity” in Fig. 4).
It is crucial to produce a certain number of offspring to maintain a sustainable population, and the very low fecundity in C. sarawakensis could be decisively disadvantageous for the species. Similar issues exist in the yolk-sac viviparous species. The yolk-sac viviparous catsharks, such as Bythaelurus clevai, B. hispidus, B. lutarius, B. stewarti and Cephalurus cephalus retain only one embryo per uterus or per mother1,28,36,37,38,KN pers.obs, which is hence termed here “single pregnancy”. These species of single pregnancy would have also lower fecundity than the species of “multiple pregnancy” seen in Galeus polli (see “Yolk-sac viviparity” in Fig. 4).
Species of the genus Cephaloscyllium are generally large in body sizes, mostly growing to more than 70 cm TL and some species (C. isabellum, C. laticeps, and C. umbratile) attain more than 100 cm TL39, whereas C. sarawakensis and C. silasi are dwarf species within the genus. Cephaloscyllium sarawakensis attains a maximum of only 39.7 cm TL in males and 49.5 cm TL in females40,present study, and matures at the sizes less than 32.5 cm TL and 35.4 cm TL in males and females, respectively41. Similarly, Cephaloscyllium silasi attains only 50 cm TL13 and reaches its maturity at less than 36.8 cm TL in males1 and less than 45 cm TL in females13. Bythaelurus clevai, B. hispidus and B. lutarius attain 42 cm TL, 36 cm TL and 39 cm TL, respectively37,39 and these species are also the smallest species for the genus. Cephalurus cephalus reaches 30 cm TL36,39, and this is known as one of the smallest sharks1.
The ratio of length at maturity to the largest total length was reported at around 0.73 for elasmobranchs42, and this indicates the smaller species could reach their maturity at smaller sizes than the larger species. Actually, the captive Cephaloscyllium umbratile which grows up to 118 cm TL hatches at lengths of 16–22 cm TL32 and attains its full maturity at 96‒104 cm TL17. In contrast, C. sarawakensis which produces very large egg cases relative to the mother size is expected to hatch at about 12 cm TL and mature at less than 35 cm TL. Therefore, C. sarawakensis grows only about 23 cm in length until maturity is attained, whereas C. umbratile needs to grow 75–90 cm to its maturity. Cephalurus cephalus produces 7–9 cm TL neonates and attains maturity at 18–22 cm TL43, thus 9–15 cm in length to grow to attain maturity. Eridacnis radcliffei gives birth 10.5‒12.8 cm TL neonates36, and attains maturity at 18.3 cm TL, only 5.5‒7.8 cm in length to the maturity.
Hence, these dwarf sharks of the sustained single oviparity and the yolk-sac viviparity of single pregnancy likely attain their maturity in a shorter time frame than the larger species do, enabling them to reproduce at an earlier age and keep their life-time fecundity high. Other factors to increase their lifetime fecundity include quick repetition of reproduction, longer lifetime reproduction and higher proportion of females, but these will not be referred to here.
Figure 6a shows five modes of lecithotrophic (yolk-dependent) reproduction in the cartilaginous fishes, and Fig. 6b1–6 denote six combinations of these reproductive modes in the catsharks. Figure 6b1 (short single oviparity) represents ten genera such as Apristurus, Asymbolus, Atelomycterus, Figaro, Haploblepharus, Holohalaelurus, Parmaturus, Poroderma, Scyliorhinus and Schroederichthys. Figure 6b3 (multiple oviparity) and Fig. 6b6 (yolk-sac viviparity of single pregnancy) are Halaelurus and Cephalurus, respectively. However, Fig. 6b2,b4,b5 include two or three modes of reproduction. Cephaloscyllium (Fig. 6b2) performs short single oviparity + sustained single oviparity, Bythaelurus (Fig. 6b4) involves short single oviparity + yolk-sac viviparity of single pregnancy, and Galeus (Fig. 6b5) performs short single oviparity + multiple oviparity + yolk-sac viviparity of multiple pregnancy. These facts could suggest rather facile diversification of reproductive mode in closely related species, or may suggest necessity of some taxonomic reconsideration of them, as indicated for Bythaelurus38.
Figure 6

Modes of reproduction (a) and combination of the modes at generic level in catsharks (b). (a) Five modes of lecithotrophy (yolk-dependent reproduction) in cartilaginous fishes, (b1) Apristurus, Asymbolus, Atelomycterus, Figaro, Haploblepharus, Holohalaelurus, Parmaturus, Poroderma, Scyliorhinus and Schroederichthys, (b2) Cephaloscyllium, (b3) Halaelurus, (b4) Bythaelurus, (b5) Galeus, and (b6) Cephalurus.

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Oviparity has been suggested to be the ancestral mode of reproduction for vertebrates7,44, and it has also traditionally been believed as the ancestral mode for chondrichthyan fishes3,14,45. However, recent studies5,6,12,46,47 suggest that viviparity is ancestral for all chondrichthyans, with many reversions to oviparity and secondary reversions to viviparity. Phylogenetic interrelationships for the Galeomorphi (orders Heterodontiformes, Orectolobiformes, Lamniformes and Carcharhiniformes)4,45,48‒50 show that short single oviparity is the ancestral mode for the Galeomorphi, and also for the orders Heterodontiformes, Orectolobiformes and Carcharhiniformes. Multiple oviparity is generally considered to have evolved from short single oviparity5, or evolved intermediately between the short single oviparity and the yolk-sac viviparity1,7,14,51.
The catsharks (now Pentanchidae and Scyliorhinidae) in the Carcharhiniformes are separated into a few isolated groups, based on genetic works49,50,52. Mapping of the reproductive modes on their phylogenetic relationships suggests that the short single oviparity is ancestral for each group. According to the phylogenetic result50 which covers more catshark taxa than the other works, their Scyliorhinidae I50 (Fig. 7a) includes nine genera of the family Pentanchidae. Six genera of these, i.e. Apristurus, Asymbolus, Figaro, Haploblepharus, Holohalaelurus and Parmaturus, display short single oviparity. The multiple oviparous genus Halaelurus is sister to the groups of short single oviparous catsharks, and the relationships suggest the multiple oviparity has derived from the short single oviparity.
Figure 7

Reproductive modes mapped on simplified relationship of (a) Scyliorhinidae I50 and (b) Bythaelurus53, and suggested evolution.

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The genus Bythaelurus, which is also deeply merged in groups of short single oviparous species in the Scyliorhinidae I50 (Fig. 7a), is currently comprised of 14 species38,53,54, with five short single oviparous species (B. bachi, B. canescens, B. dawsoni, B. naylori and B. vivaldi) and four yolk-sac viviparous species of single pregnancy (B. clevai, B. hispidus, B. lutarius and B. stewarti) (Fig. 6b4). Interrelationships of five Bythaelurus species53 (Fig. 7b) show they are clearly separable in two groups, i.e. short single oviparous species (B. bachi, B. naylori, B. dawsoni and B. canescens) and yolk-sac viviparous species of single pregnancy (B. hispidus). The short single oviparous B. dawsoni and B. canescens have a sister relation with short single oviparous genera Asymbolus + Figaro in the Scyliorhinidae I50 (Fig. 7a). These facts suggest that the short single oviparity is ancestral for Bythaelurus and the yolk-sac viviparity of single pregnancy could have derived from the short single oviparity1, maybe via sustained single oviparity.
The genus Galeus contains 18 species with three reproductive modes (Fig. 6b5), i.e. short single oviparity (G. antillensis and seven other species), multiple oviparity (G. atlanticus, G. melastomus and G. piperatus) and yolk-sac viviparity of multiple pregnancy (G. polli). The genus Galeus is deeply embedded within groups of short single oviparous species in the Scyliorhinidae I50 (Fig. 7a), and the short single oviparity is considered to be ancestral for the genus Galeus, and the yolk-sac viviparity of multiple pregnancy in G. polli could have derived from short single oviparity via multiple oviparity.
Their Scyliorhinidae II50 includes three genera Atelomycterus, Schroederichthys of short single oviparity and oviparous Aulohalaelurus55. Scyliorhinidae III50 includes three genera Cephaloscyllium, Scyliorhinus and Poroderma, and they all display short single oviparity. Cephaloscyllium sarawakensis and C. silasi of sustained single oviparity were not treated in their analysis50, but the relationships of Cephaloscyllium in the Scyliorhinidae III50 that is composed of short single oviparous species could suggest derivation of the sustained single oviparity directly from short single oviparity by longer retention of one egg case in an oviduct.
The modes of reproduction in the catsharks were summarized in Table 2, and the phylogenetic evidences mentioned above suggest: (1) short single oviparity is ancestral for the catsharks; (2) more diverse modes of reproduction evolved in the family Pentanchidae than family Scyliorhinidae; (3) sustained single oviparity in Cephaloscyllium was derived directly from short single oviparity; (4) multiple oviparity in Halaelurus was derived from short single oviparity; (5) multiple oviparity in Galeus was derived from short single oviparity, and originated yolk-sac viviparity of multiple pregnancy; (6) yolk-sac viviparity of single pregnancy in Bythaelurus was derived from short single oviparity, possibly via sustained single oviparity; and (7) yolk-sac viviparity of single pregnancy in Cephalurus was derived possibly from short single oviparity via sustained single oviparity.
Table 2 Modes of reproduction and evolution in catsharks.
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Materials and methods
All specimens of Cephaloscyllium sarawakensis examined were bycatch from commercial bottom trawlers operating in the South China Sea off southwest Taiwan, and were collected at Hsin-da port (HD) and Ke-tzu-liao (KTL) in Kaohsiung. Specimens were fixed in 4% formalin and then transferred to 70% Ethanol or 50% Isopropanol ethanol. All specimens were deposited at Pisces collection of the National Museum of Marine Biology & Aquarium, Pingtung, Taiwan (NMMB-P). Total length (TL) was measured using a ruler or digital caliper, to nearest 1 or 0.1 mm, respectively.
Egg cases (Table 3): a total of 8 egg cases without visible embryo on the yolk, and 15 egg cases with a developed embryo each were collected from the oviduct of thirteen specimens of C. sarawakensis. Two egg cases with a developed embryo tied on the tube of a tubeworm Paradiopatra sp. (family Onuphidae, order Eunicida) were collected from the fishery landings by fishers, and were kept frozen. Egg cases were deposited and catalogued in NMMB-P collection. Length (excluding tendrils, ECL) and width (ECW) of the egg case were measured by a ruler or digital caliper.
Table 3 Measurements of egg cases and embryos, and sampling data of Cephaloscyllium sarawakensis.
Full size table

Juveniles: NMMB-P22719, 125 mm TL female, KTL, 2 Apr. 2015; NMMB-P17143, 143 mm TL male; NMMB-P24872 (1 of 6 specimens), 134 mm TL male, KTL, 18 Mar. 2016. More

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Ethic statement
All animal experiments were performed in compliance with the German animal protection law (TierSchG) and approved by the local animal welfare committees, the Landesamt für Natur, Umwelt und Verbraucherschutz, North Rhine Westfalia (84–02.04.2016.A207 and 84–02.04.2015.A293). All C57BL6J wildtype mice were bred locally and held under specific pathogen-free conditions at the Institute of Laboratory Animal Science at RWTH Aachen University Hospital. The day of birth was considered day 0, i.e., animals screened at day 1 were approximately 24 h old and verified to have ingested breast milk (abdominal milk spot). Mice were weaned at PND21.
In vivo study design
To monitor microbiota and host metabolic development throughout the neonatal period into adulthood, intestinal and hepatic tissues were obtained from C57BL/6 J mice in two different approaches. First, total small intestinal, colon, and liver tissues were obtained from C57BL/6 J mice aged 1, 7, 14, 21, 28, and 56 days (n = 5 per timepoint). In a separate set of experiments, similar tissues were obtained from mice aged 0, 6, 12, 18, and 24 h (n = 10 per timepoint). To rule out potential litter and cage effects, we obtained tissues from a single animal of one litter for a given age group and repeated this by selecting a new animal from the same litter for every other age group (Fig. 1a). Every animal was only examined once at the indicated age (PND). This means that in total 30 animals from 5 litters were used for monitoring the microbiota development between age 1 and 56 days and 46 animals from 10 litters to monitor the microbiota development within the first 24 h. For oral bile acid administration, 7–14 (UDCA and Control, n = 14;, GCA, n = 11; TCA, n = 10; βTMCA, n = 7) PND7 animals received the indicated bile acid (Sigma-Aldrich, Biomol) at a concentration of 70 µg/g body weight or PBS daily for 3 days by oral gavage (average 5 µL) with 100% succession47. Body weight and food/water consumption was monitored daily. To determine the effect of bile acid administration, the intestine was aseptically cut into 10–20 parts and alternately assigned to two collections for microbial profiling and assessment of metabolites. Samples from an additional group of adult 8–12 week old animals (n = 5) were processed in parallel to provide an adult microbiota control. Tissues were transferred into sterile micro-centrifuge tubes and stored at −80 °C before analysis. Liver tissues of 7-, 14-, 21- and 56-day-old germ-free mice were obtained from the Institute for Laboratory Animal Science at Hannover Medical School. Tissues were collected and stored in sterile tubes stored at −80 °C until further analysis.
DNA isolation and generation of sequence data
Total metagenomic DNA was isolated from snap frozen small intestinal and colonic tissues by repeated-bead-beating (RBB) combined with chemical lysis plus a column-based purification method48. Approximately 200 mg tissue was added to a 2.0 mL screw-cap tube containing 0.5 g of 0.1 mm zirconia beads (Biospec Products, Bartlesville, OK, USA) and 1 mL ASL lysis buffer from the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). Samples were incubated at 15 min. at 95 °C and subsequently two successive rounds of bead-beating were employed using a FastPrep®−24 instrument (MP Biomedicals, Inc., USA) at 5.5 ms−1 (3 × 1 min. for each round). To minimize physical damage of DNA in the RBB method, the lysate fraction produced from the first round of bead beating was drawn after centrifugation at full speed (~160,000 × g) for 5 min at 4 °C. A second round of bead beating was performed upon adding 300 μL fresh lysate buffer after which supernatants were pooled. To precipitate nucleic acids, 260 μL 10 M ammonium acetate was added to the lysate tubes, mixed and incubated on ice for 5 min. After centrifugation at 4 °C for 15 min. at full speed, supernatants were transferred to a new 1.5 mL tube to which an equal volume of isopropanol was added. Next, samples were incubated on ice for 30 min., centrifuged at RT for 15. min. at full speed and supernatants were removed by decanting. The nucleic acid pellet was washed with 500 μL 70% EtOH and dried under vacuum for 3 min. The nucleic acid pellet was dissolved in 100 μL TE (Tris-EDTA) buffer. Two microliter DNase-free RNase (1 mg/mL) was added and samples were incubated at 37 °C for 15 min. Next, 15 μL proteinase K and 200 μL AL buffer were added, samples were vortexed for 15 s. and incubated at 70 °C for 10 min. After adding 200 μL ethanol (96–100%) and vortexing, the lysate was transferred to QIAamp spin columns (Qiagen, Hilden, Germany). The DNA was finally purified using the QIAamp DNA Stool Mini Kit according to the manufacturer’s instructions and eluated in 200 μL AE Buffer. For each DNA isolation batch, additional isolation was performed on PCR-grade water as a negative control. Generation of amplicon libraries and sequencing was performed according to a previously published protocol49. Briefly, the V4 region of the 16S rRNA gene was PCR amplified from each DNA sample in duplicate in 50 μL volumes containing 10 pmol of both primers (5′-GTGCCAGCMGCCGCGGTAA*-3′ [515 F] and barcoded 5′-GGACTACHVGGGTWTCTAAT*-3′ [806 R]), 5 μL Accuprime buffer II, 0.2 μL Accuprime Hifi polymerase (Thermo Fisher Scientific, Waltham, WA, USA) and 2 μL DNA. After an initial denaturation step at 94 °C for 3 min., amplification was carried out for 30 s. at 94 °C, 45 s at 50 °C and 1 min. at 72 °C. Amplification was carried out in 35 cycles for PND1-PND56; the samples with a low yield of DNA required 40 cycles (0–24 h) The PCR program ended with a final post-PCR incubation step of 10 min at 72 °C to promote complete synthesis of all PCR products. Pooled amplicons from the duplicate reactions were purified using AMPure XP purification (Agencourt, Massachusetts, USA) according to the manufacturer’s instructions and subsequently quantified by Quant-iT PicoGreen dsDNA reagent kit (Invitrogen, New York, USA). Amplicons were mixed in equimolar concentrations, to ensure equal representation of each sample, and sequenced on an Illumina MiSeq instrument using the V3 reagent kit (2 × 250 cycles). All V4 16S rDNA bacterial sequences generated in this study have been submitted to the Qiita and ENA databases under accession No. 10719 and ERP116798, respectively.
Sequence analysis
Data demultiplexing, length and quality filtering, pairing of reads and clustering of reads into Operational Taxonomic Units (OTUs) at 97% sequence identity was performed using the online Integrated Microbial Next Generation Sequencing (IMNGS, www.imngs.org) platform using default settings50. Removal of primers and technical reads resulted in fragments of approximately 250 bases. Sequencing was performed from both the 3′ and 5′ side resulting in sufficient resolution. IMNGS is a UPARSE based analysis pipeline51. Pairing, quality filtering and OTU clustering (97% identity) was done by USEARCH 8.052. The analysis was based on OTUs rather than amplicon sequence variants (ASVs) since we aimed at aggregating taxa at a higher level and wanted to avoid overestimation of prokaryotic diversity due to Intragenomic heterogeneity of 16S rRNA genes53. Chimera filtering was performed by UCHIME (with RDP set 15 as a reference database54. Taxonomic classification was done by RDP classifier version 2.11 training set 15.8 Sequence alignment was performed by MUSCLE and treeing by Fasttree55,56. A total of 21,372,397 V4 reads were generated over two runs. After trimming, quality filtering, removal of potential chimeric reads, de-multiplexing and removal of low abundant operational taxonomic units (OTUs), 15,692,587 sequences belonging to 478 OTUs were retained for downstream analysis. Negative controls were evaluated based on their number of sequences and composition compared to other samples. We used for each batch, sampling blank controls, DNA blank extraction controls and no-template amplification controls, and monitored the lack of contaminant bacterial DNA load herein by a gel-based principle. Moreover, we compared the acquired OTUs composition from the negative controls to our low abundant microbial samples to ensure that our findings were not driven by potential contaminant taxa. Subsequently, samples of the negative controls and with low sequencing depth (less than 6,749 sequences/sample) were excluded from subsequent analysis. For the remaining samples, the number of sequences per sample ranged from 6749 to 239,395 (median 87,227).
Richness, diversity, taxonomy, and enterotype analyses
Data normalization, diversity, taxonomical binning and group comparisons were performed using the Rhea package version 1.657. In order to not discard informative data, normalization in Rhea was performed by dividing OTU counts per sample for their total count (sample depth) followed by multiplying all of the obtained relative abundance for the lowest sample depth (6749 reads/sample). Alpha- (observed species and Shannon index) and the generalized Unifrac beta-diversity index were calculated using the Rhea package58. Additional beta-diversity indices (weighted Unifrac, unweighted Unifrac, and Bray–Curtis distance) were calculated using the R package (version 3.6.1.) Phyloseq package version 1.30.059. Ordination of samples according to their microbial composition expressed as Hellinger transformed genus abundance data or beta-diversity indices was visualized using Principal Component Analysis (PCA) and Principal Coordinate Analysis (PCoA), respectively. All ordinations were constructed using the R package Phyloseq and included 95% confidence ellipses. Dirichlet multinomial mixtures models (DMM) were used to calculate genus-level enterotypes60. When including samples from all time-points, Laplace approximation revealed an optimal number of three clusters.
Downstream microbial analyses and presentation
Smoothing of the kinetic for dominant taxa (Fig. 1e and f, as well as Supplementary Fig. 1d and e) was generated using the geom_smooth function of the ggplot package 3.2.1. with default settings. The lines reflect the mean values of the relative abundance. The appearance and disappearance of OTUs of the dominant phyla (Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes) with age was visualized in Sankey-plots (SankeyMATIC.com). For readability of Sankey-plots, only OTUs present in >10% of all samples per timepoint and with a prevalence of >20% in the entire dataset, were included. Ecosystem specific functional metagenome predictions were created by the novel PICRUSt-iMGMC workflow (using PICRUSt version 1.1.3) with the de novo picked OTUs and using mouse metagenome-assembled genomes linked to 16S rRNA genes61. The derived KEGG orthologs were mapped into multiple pathways or modules. Differentially changed KEGG modules were identified using the pathways enrichment analyses from MicrobiomeAnalyst with default settings62. The identification of lactobacillus-related OTUs were performed using EZbiocloud and used to construct a phylogentic tree of lactobacilli by MEGA7 (MUSCLE) for alignment and iTOL v4 for the final annotations (itol.embl.de) with default settings.
Liver metabolomics and bile acid analyses
The metabolome analyses were carried out with the AbsoluteIDQ® p180 Kit (Biocrates Life Science AG, Austria. The kit allowed identification and quantification of 188 metabolites from 5 compound classes (acyl carnitines, amino acids, glycerophospho-, and sphingolipids, biogenic amines, and hexoses). The kit used flow injection tandem mass spectrometry (FIA) for the non-polar metabolites and LC-MS/MS for the more polar compounds. The integrated MetIDQ Software (version Boron 2623) streamlined the data analysis by automated calculation of metabolite concentrations. Quantification of analytes utilized stable isotope-labeled or chemically homologous internal standards (IS). Controls were included for 3 different concentration levels. For calibration, the kit contained a calibrator mix at 7 different concentrations. The measurements were carried out with an ABI Sciex API5500 Q-TRAP mass spectrometer via Electrospray ionization (ESI) by Multi Reaction Monitoring (MRM) mode for high specifity and sensitivity. 158 MRM pairs were measured in positive ion mode (13 IS) and 2 MRM pairs were measured in negative mode (1 IS). The following additional chemicals for LC-MS were used: water, Millipore; PITC, Fluka; pyridine, Fluka (p.a.); methanol, Merck; Lichrosolv for LC/MS; acetonitrile, Merck; formic acid, Sigma Aldrich. Metabolites were extracted from liver samples by adding H2O/acetonitrile (1:1,v:v) per mg sample followed by homogenization with a tissue disruptor (10 min, 30 Hz, 4 steel balls). The samples were centrifuged (1400×g, 2 min) and the supernatant analyzed. The targeted analysis was performed by adding 10 μL of extracted liver sample to the AbsoluteIDQ® p180 Kit (Biocrates Life Science AG, Innsbruck, Austria), following the vendor’s instructions63. For bile acid measurements the MS-based Bile Acids Kit (Biocrates Life Sciences AG, Innsbruck, Austria), a 96-well plate format assay, was used following the manufacturer´s instructions with normalization based on mass64. The following settings were used: turbo spray for ion source, 20 for Curtian Gas, medium for CAD Gas, 40 psi for ion source gas 1 and 50 psi for ion source gas 2. For the bile acid measurements we used an ion spray voltage of −4500 V and a temperature of 600 °C; for the p180 kit we used an ion spray voltage of 5500 V and a temperature of 500 °C. All metabolomic data generated in this study have been submitted to the Metabolomics Workbench and have been assigned the Study ID ST001388, ST001389, ST001396, ST001397, the Project ID PR000952 and the Project DOI 10.21228/M8N397.
The contribution of each metabolite to metabolomic variation was derived from age-constrained redundancy analysis (RDA) based on all metabolites stratified in group levels with additional scaling by normalization based on z-scores (Phyloseq package). Moreover, PCA was used to illustrate changes in the composition of the different metabolic groups during the postnatal period for both PC1 and PC2, and PC2 and PC3. For the bile acids the 2nd and 3rd component were chosen for plotting, since the first component was mainly driven by the strong separation observed between the first and subsequent time points (PND1 vs. PND7–56).
Multi-omics analyses
Regularized canonical correlation analyses (rCCA) were performed (Mixomics package 6.10.8)65 to unravel specific correlations between bile acids and OTUs with a minimal presence of 20% in all samples. Samples were excluded from the microbiota-data if they were not measured in the bile acid analyses (i.e. PND1 of litter 1 and 2). Prior to rCCA a hyperbolic sine transformation was used on OTU-counts and a log-transformation for the bile acids. For the estimation of regularization (penalization) parameters λ1 and λ2, the cross-validation procedure (CV) method was used. We used a λ1 = 0.0001, λ2 = 1 with a CV-score = 0.4779644 and 2 components. OTUs with a correlation between −0.3 and 0.3 on the first 2 components were filtered out to optimize the rCCA.
The Spearman’s rank correlation coefficient was calculated between bile acids (weight corrected) and bacterial genera (relative abundances) with a minimal presence of 20% in all samples. Benjamini and Hochberg FDR correction was performed to correct for multiple testing (p  > 0.05). For the heatmap only significant correlations with adjusted p-value of More