Ecology

Site and sampling
We selected twelve monoculture tree stands of the most common tree species in Britain, Scots pine (Pinus sylvestris L.), Sitka spruce (Picea sitchensis Bong. Carr.), pedunculate oak (Quercus robur L.) and common beech (Fagus sylvatica L.). The majority of the stands were experimental sites within the Level II- ICP intensive forest monitoring network (http://icp-forests.net/), with the exception of Covert Wood, Shobdon and Goyt. The Goyt site was added as a high Ndep site as a contrast to the low Ndep Sitka spruce site in Scotland (Fig. 1, Table 1, Supplementary Table 1). For each species, forests were selected with similar soil type and age, but with contrasting Ndep, Sdep and climate, particularly rainfall and temperature, as described in Fig. 1, Table 1 and Supplementary Table 1. Stand information (mean tree height, mean diameter at the breast height and basal area) as measured for target years and for some of the forest stands are shown in Fig. S4.
At each ICP forest site, a plot of 0.25 ha was established in 1995 to carry out monitoring30 and a similar protocol was followed at the Goyt and Shobdon sites. Within each plot, 10 trees were selected for the collection of 3 wood cores per tree by using a 5 mm diameter increment borer, which were placed in paper straws for transport. Sampling was carried out between November 2010 and March 2011. Once in the laboratory, samples were dried at 70 °C for 48 h. Of the three wood cores sampled, one was kept for dendrochronology, with the other two used for stable isotope analyses.
Climate and atmospheric deposition data
Climate data (Temperature, T, Vapour Pressure Deficit, VPD, Precipitation, P) were obtained from automated weather stations at the sites and/or the nearest local meteorological stations (data were provided by the British Atmospheric Data Centre). Annual mean (Ta) and mean maximum (Tamax) values for temperature were calculated from monthly mean and maximum air temperature, T, respectively, and annual precipitation (Pa) was calculated as the sum of total monthly precipitations. Annual VPD was calculated from averaging monthly values obtained from mean monthly maximum temperature and minimum monthly relative humidity. For all the parameters, mean values were also calculated over the growing season, i.e., from May to September. We also considered the standardised precipitation-evapo-transpiration index, SPEI, relative to August, with 1 (SPEI8_1), 2 (SPEI8_2) and 3 (SPEI8_3) months time-scale and SPEI relative to December, with 1 and 12 months time-scale, the latter providing year-cumulated soil moisture conditions. SPEI values were obtained from the global database with 0.5 degrees spatial resolution available online at: https://sac.csic.es/spei/.
Yearly wet nitrogen (Ndep) and sulphur deposition (Sdep) were obtained from measured bulk precipitation and throughfall water volumes at the sites and measured elemental concentrations (NO3−, NH4+ and SO2–4) as previously described30. For the spatial analyses, we considered mean of annual deposition (sNdep and sSdep), obtained as the sum of total (NH4-N + NO3-N for Ndep) wet and dry deposition. The latter were estimated as difference between throughfall and bulk precipitation N fluxes30. For Rogate only 1 year (2010) of monitoring was available. For Goyt site, atmospheric deposition data collected at Ladybower were considered, as the two sites are 30 km apart. For two sites (i.e., Shobdon and Covert Wood), which were not part of the regular ICP forest sites, the wet deposition obtained from the UK 5 × 5 km grid Ndep and Sdep dataset was used4. The estimate included wet and dry NHx-N (NH4, NH3), NOy-N (NO2, NO3, HNO3) and S (SOx = SO2 and SO4) deposition, modelled using FRAME with 2005 emissions data4. However, only the total wet deposition was included in the analyses here, as we previously reported a good agreement between modelled and measured wet Ndep50.
For the temporal analyses, only wet deposition (as calculated from NO3−, NH4+ and SO2–4 concentrations in bulk precipitation) was considered (indicated as aNdep and aSdep), given the uncertainties associated with the quantification of the dry deposition. For instance, when differences between throughfall and bulk precipitation are  More

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The Aedes aegypti mosquito’s taste for human blood has been linked to exposure to dense settlements dotted with sources of water. Credit: Getty

Ecology
23 July 2020

Population density and water storage created a new ecological niche for the disease-carrying insects.

As humans crowd into cities where standing water abounds, even in the driest months, we become an ever-more-tempting target for bloodsuckers.
Only a few of the more than 3,000 species of mosquito specifically seek out blood from humans — but those few are enough to spread diseases that affect some 100 million people every year. To shed light on the evolutionary origin of insects’ taste for humans, Lindy McBride and Noah Rose at Princeton University in New Jersey and their colleagues captured Aedes aegypti mosquitoes at 27 sites across the insects’ ancestral range in sub-Saharan Africa.
The researchers placed hungry females in boxes with two exits: one offering the smell of a human and the other the smell of either a living guinea pig (Cavia porcellus) or a button quail (Coturnix coturnix). Compared with mosquitoes that were indifferent to human blood, those that sought it out were more likely to hail from areas with dense human populations and long, hot, dry seasons.
The researchers suspect that water stored by humans has become a key breeding location in a landscape with little standing water. Their genetic analysis suggests that the taste for humans evolved just once. More

Study site
The study was conducted in the Federseemoor (48.092°N, 9.636°E), a peatland of 30 km2 located in the region Upper Swabia in southwest Germany. This peatland has developed via natural terrestrialization from a proglacial lake after the last ice age. As a result, the surface area of the lake declined from 30 to 12 km2. Between 1787 and 1808, the lake was further reduced to a size of 1.4 km2 by drainage activities. The newly gained land of 11 km2 was used as pasture but turned out to be unprofitable due to the recurring high water table. Nowadays it is a nature conservation area, mainly consisting of fen (see van den Berg et al.21 for a vegetation map). The lake Federsee is completely surrounded by reed vegetation (P. australis), with a total area of 2.2 km2 and a density of around 70 living shoots and 75 dead stems per m2. During the measurement period (7–10 June) the Phragmites plants were 1.2 m high. This is half their maximum height, which is reached at the end of July. The high density of Phragmites and lack of other species in the reed belt result from high nutrient concentrations due to wastewater input to the lake since 1951. After 1982, the input of untreated sewage water was stopped, which reduced the nutrient concentrations. Only since 2006 has there been a significant improvement in water quality, and after 2008 the lake water became clear again. The field experiment was installed in the middle of the reed area at around 70 m distance from an eddy covariance (EC) tower, which has been running since March 201321. In a radius of at least 200 m around the EC tower, the vegetation is dominated by Phragmites (see van den Berg et al.21), meaning only reed dominated the measured EC footprint.
Field experiment
Nine plots of 2 m × 2 m were prepared for three treatments with three replicates: (1) clipped reed (CR), to exclude the pressurized flow in the plants; (2) clipped and sealed reed (CSR), to exclude any exchange via plant stems; and (3) control where reed was not manipulated. In the CR and CSR treatments, living and dead reed stems were clipped to about 10 cm above the water table. In the CSR treatment the clipped reed stems was sealed with an acrylic sealant. Since rhizomes connect plants over longer distances, plots were isolated by cutting rhizomes from the reed plants around each plot to a depth of 50 cm, to avoid gas exchange with the surrounding area. The period between preparation of the plots and measurements was minimized (1–2 days) to reduce possible side effects, such as change in substrate availability for methanogens. One day before the first measurement, the water table rose about 20 cm in the whole field, flooding the prepared sealed stems of one plot already prepared for the CSR treatment. Nevertheless, since no gas exchange is expected from the sealed stems, this plot was still included in the experiment. CH4 and CO2 diffusive fluxes from the soil and plant-mediated fluxes were measured with transparent flow through chambers. Pore water was extracted to analyze the effect of the reduced/excluded gas exchange by the plants on soil chemistry. In each plot ebullition was measured as well (see below).
Diffusive and plant mediated CH4 flux
On 7, 9 and 10 June 2016 between 07:00 and 18:00, the gas fluxes of each treatment were alternately measured. Per day, only one of the triplicates per treatment was measured. CH4 fluxes were measured in the middle of the plots with transparent chambers with a diameter of 50 cm. One chamber was 2 m high and was on the control plots. Two chambers were 1 m high and used on the CR and CSR plots. The 1-m chambers were equipped with a small fan of 8 cm × 8 cm that had a flow capacity of 850 l min−1; two fans were installed in the 2-m chamber. Each day one replicate of every treatment was measured, to be able to capture the diurnal cycle for each plot and to minimize disturbance by translocating the chambers. The chambers were connected with 8 m tubing to a multiport inlet unit attached to a fast greenhouse gas analyzer (GGA) with off-axis integrated cavity output spectroscopy (GGA-24EP, Los Gatos Research, USA) measuring the concentration of CH4 and CO2 every second. Every 5 min, the multiport switched between the three chambers, allowing air from each chamber to be alternately pumped through the GGA with a pumping rate of 300 ml min−1 and resulting in four flux measurements per plot per hour (~ 35 measurements per plot per day). The withdrawn air from the chamber was replaced with ambient air through an opening in the chamber. After 1–2 h of continuous measurements, the chambers were ventilated by lifting the chambers to fully replace inside air with ambient air. After 15 min, the chamber was put back and measurements continued. Since it takes a long time before the chamber CH4 gets to equilibrium with the water column, 1–2 h of increasing CH4 concentration in the chamber will have little effect on the measurement accuracy of the CH4 flux (in contrary to the CO2 flux)22. Nevertheless, we used only data from the first 30 min after ventilating to calculate the diffusive flux (five measurements per plot per day), since this is the period where temperature and humidity inside the chamber resemble outside conditions most closely. Only for the comparison between eddy covariance fluxes and chamber fluxes on the control plots we did use data from the whole measurement period.
The concentration for every measurement point was corrected for the change in concentration caused by the inflow of ambient air with known CO2 and CH4 concentrations (measured by the EC station) and outflow of chamber air (both with a flow rate of the pump speed of the Los Gatos). The slope of the corrected chamber concentrations over a 4 min period within the 5 min measurement was used to calculate the flux and was checked for non-linear fluctuations due to e.g. ebullition. Fluxes corresponding to an average chamber concentration of  > 100 ppm CH4 were discarded, because of the GGA’s detection limit. In total 11% of the fluxes were discarded.
Ebullition
In each plot ebullition was measured by catching bubbles from a fixed surface with an ebullition trap10, composed of a 20 cm diameter funnel, to which a glass bottle of 300 ml was attached. The bottles were filled with water from the site and the ebullition trap was installed under the water table on 8 June and carefully anchored between reed stems (no open endings of stems were below the trap) on the soil surface around 0.55 m below the water surface. Bubbles were captured in the glass bottle for 18 days, after which the bottles were removed and gas samples were taken in the field. The total volume of ebullition gas was determined and the concentration of CH4, CO2 and N2O were measured by gas chromatography (7890B GC, Agilent Technologies, USA) in the lab.
Environmental variables
In each chamber, temperature and radiation were measured with a temperature/light sensor (HOBO Pendant data logger, Onset Computer Corporation, USA) logging at an interval of 30 s. Every minute soil temperature was measured in each plot in the upper 0–0.05 m with a Soil Water Content Reflectometer (CS655, Campbell Scientific Inc., USA) around 0.56 m below the water table. Air temperature, air relative humidity (HMP155, Vaisala Inc., Finland) and incoming and outgoing shortwave and longwave radiation (CNR4, Kipp & Zonen Inc., The Netherlands) were measured at a height of 6 m close to or at the EC station. Groundwater table was continuously measured with a water level pressure sensor (Mini-Diver datalogger, Eijkelkamp Agrisearch Equipment Inc., The Netherlands) placed at 1.45 m depth in a 2-m long filter pipe that was placed 1.60 m into the soil. Data were recorded at a 30 min interval.
Pore water sampling and analysis
To see if the treatments had any effect on the methane production, pore water samples were analyzed. At two locations in each plot, pore water was extracted anaerobically with ceramic cups (Eijkelkamp Agrisearch Equipment Inc., The Netherlands). Pore water from 10, 20, 30 and 50 cm depth was collected by vacuum suction in syringes and transported to the lab. In the lab, pore water was diluted with a ratio of 1:3. Dissolved organic carbon (DOC) concentration was measured with a Dimatoc 100 DOC/TN-analyzer (Dimatec, Germany). A second pore water sample was taken in vacuumed 13 ml exetainers with 3 g of NaCl. The concentration of CH4 in the headspace of these exetainers, representing the CH4 concentration in pore water, was determined on a HP gas chromatograph (Hewlett Packard, USA). A third pore water sample was fixed with 0.2% 2.2-bipyridin in 10% CH3COOH buffer in the field to determine Fe(II) measuring photometrical absorption at 546 nm in the lab.
Eddy covariance
The EC tower was located at a distance of around 70 m from the prepared plots. The tower was 6 m high and consisted of a LI-7700 open path CH4 gas analyser (LI-COR Inc., USA), a LI-7200 enclosed path CO2/H2O gas analyser (LI-COR Inc., USA) and a WindMaster Pro sonic anemometer (GILL Instruments Limited Inc., UK). Molar mixing ratio/mass density of the gases and wind speed in three directions were measured at a frequency of 10 Hz. Fluxes were calculated for an averaging interval of 15 min with the software EddyPro version 6.1.0. For more detailed information about the set up and calculations of the fluxes, see van den Berg et al.21.
δ13C measurements
CH4 oxidation and transport lead to isotopic fractionation of δ13C of CH423. The difference between δ13C of the CH4 present in the soil and the CH4 emitted to the atmosphere may therefore reveal the importance of both methane oxidation and the different emission pathways.
The δ13C of CH4 tends to be much lower than the natural abundance in organic compounds, because methanotrophic prokaryotes prefer the lighter 12CH4 to 13CH4 thereby increasing the δ13C of CH4. Diffusion rates for 12CH4 are higher than for 13CH414 decreasing the δ13C of the emitted CH423. Although 13C enrichment (compared to produced CH4) has been found in internal spaces of plants due to CH4 oxidation14, the fractionation at the plant-atmosphere surface reduces the δ13C by about 12–18‰ due to the faster transport rate of 12CH4, which makes that emitted CH4 can have a lower fraction of δ13C than the produced CH4. Differences in δ13C between sediment and overall emission are larger for plants with diffusive internal gas transport than for plants with convective gas transport23.
Since fractionation of CH4 emitted through ebullition in shallow waters is negligible, these gas bubbles can be used to know the isotopic composition of CH4 produced in sediment23. We therefore compared the δ13CH4 signature of ebullition gas with the signatures of CH4 from the chambers. Gas samples from the chamber were taken when the CH4 concentration was at least 10 times the ambient concentration, from each plot in the afternoon. The δ13CH4 signature was measured with an isotope-ratio mass spectrometer Delta plus XP (Thermo Finnigan, Germany).
Statistics
Chamber fluxes were measured at different times of the day, which means that environmental variables like temperature and radiation were varying. To be able to compare the different treatments without the variation resulting from environmental conditions, an analysis of covariance (ANCOVA) was conducted with the environmental variables as covariables. For the analysis, the data of the different measurement days were pooled together per treatment. The residuals of the model were normally distributed. With the parameters of the ANCOVA model, average fluxes were calculated with average environmental variables for the period ebullition was measured (8–27 June), to be able to compare the chamber fluxes with ebullition.
To test if the means of the ebullition measurements or pore water concentrations were different between the treatments, an analysis of variance (ANOVA) test was performed with Fishers’s Least Significant Difference (LSD) post hoc test to find the specific differences between the treatments. More

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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.
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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