Universal target‐enrichment baits for anthozoan (Cnidaria) phylogenomics: New approaches to long‐standing problems

Anthozoans (e.g., corals, anemones) are an ecologically important and diverse group of marine metazoans that occur from shallow to deep waters worldwide. However, our understanding of the evolutionary relationships among the ~7,500 species within this class is hindered by the lack of phylogenetically informative markers that can be reliably sequenced across a diversity of taxa. We designed and tested 16,306 RNA baits to capture 720 ultraconserved element loci and 1,071 exon loci. Library preparation and target enrichment were performed on 33 taxa from all orders within the class Anthozoa. Following Illumina sequencing and Trinity assembly, we recovered 1,774 of 1,791 targeted loci. The mean number of loci recovered from each species was 638 ± 222, with more loci recovered from octocorals (783 ± 138 loci) than hexacorals (475 ± 187 loci). Parsimony informative sites ranged from 26 to 49% for alignments at differing hierarchical taxonomic levels (e.g., Anthozoa, Octocorallia, Hexacorallia). The per cent of variable sites within each of three genera (Acropora, Alcyonium, and Sinularia) for which multiple species were sequenced ranged from 4.7% to 30%. Maximum‐likelihood analyses recovered highly resolved trees with topologies matching those supported by other studies, including the monophyly of the order Scleractinia. Our results demonstrate the utility of this target‐enrichment approach to resolve phylogenetic relationships from relatively old to recent divergences. Redesigning the baits with improved affinities to capture loci within each subclass will provide a valuable toolset to address systematic questions, further our understanding of the timing of diversifications and help resolve long‐standing controversial relationships in the class Anthozoa.     

Transcriptome‐based target‐enrichment baits for stony corals (Cnidaria: Anthozoa: Scleractinia)

Despite the ecological and economic significance of stony corals (Scleractinia), a robust understanding of their phylogeny remains elusive due to patchy taxonomic and genetic sampling, as well as the limited availability of informative markers. To increase the number of genetic loci available for phylogenomic analyses in Scleractinia, we designed 15,919 DNA enrichment baits targeting 605 orthogroups (mean 565 ± SD 366 bp) over 1,139 exon regions. A further 236 and 62 barcoding baits were designed for COI and histone H3 genes respectively for quality and contamination checks. Hybrid capture using these baits was performed on 18 coral species spanning the presently understood scleractinian phylogeny, with two corallimorpharians as outgroup. On average, 74% of all loci targeted were successfully captured for each species. Barcoding baits were matched unambiguously to their respective samples and revealed low levels of cross‐contamination in accordance with expectation. We put the data through a series of stringent filtering steps to ensure only scleractinian and phylogenetically informative loci were retained, and the final probe set comprised 13,479 baits, targeting 452 loci (mean 531 ± SD 307 bp) across 865 exon regions. Maximum likelihood, Bayesian and species tree analyses recovered maximally supported, topologically congruent trees consistent with previous phylogenomic reconstructions. The phylogenomic method presented here allows for consistent capture of orthologous loci among divergent coral taxa, facilitating the pooling of data from different studies and increasing the phylogenetic sampling of scleractinians in the future.     

Nest attendance by tropical and temperate passerine birds: Same constancy,different strategy

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The influence of locus number and information content on species delimitation: an empirical test case in an endangered Mexican salamander

Perhaps the most important recent advance in species delimitation has been the development of model‐based approaches to objectively diagnose species diversity from genetic data. Additionally, the growing accessibility of next‐generation sequence data sets provides powerful insights into genome‐wide patterns of divergence during speciation. However, applying complex models to large data sets is time‐consuming and computationally costly, requiring careful consideration of the influence of both individual and population sampling, as well as the number and informativeness of loci on species delimitation conclusions. Here, we investigated how locus number and information content affect species delimitation results for an endangered Mexican salamander species, Ambystoma ordinarium. We compared results for an eight‐locus, 137‐individual data set and an 89‐locus, seven‐individual data set. For both data sets, we used species discovery methods to define delimitation models and species validation methods to rigorously test these hypotheses. We also used integrated demographic model selection tools to choose among delimitation models, while accounting for gene flow. Our results indicate that while cryptic lineages may be delimited with relatively few loci, sampling larger numbers of loci may be required to ensure that enough informative loci are available to accurately identify and validate shallow‐scale divergences. These analyses highlight the importance of striking a balance between dense sampling of loci and individuals, particularly in shallowly diverged lineages. They also suggest the presence of a currently unrecognized, endangered species in the western part of A. ordinarium's range.     

Phylogenomic species delimitation and host‐symbiont coevolution in the fungus‐farming ant genus Sericomyrmex Mayr (Hymenoptera: Formicidae): ultraconserved elements (UCEs) resolve a recent radiation

Ants in the Neotropical genus Sericomyrmex Mayr cultivate fungi for food. Both ants and fungi are obligate, coevolved symbionts. The taxonomy of Sericomyrmex is problematic because the morphology of the worker caste is generally homogeneous across all of the species within the genus, species limits are vague, and the relationships between them are unknown. We used ultraconserved elements (UCEs) as genome‐scale markers to reconstruct evolutionary history and to infer species boundaries in Sericomyrmex. We recovered an average of ～990 UCE loci for 88 Sericomyrmex samples from across the geographical range of the genus as well as for five outgroup taxa. Using maximum likelihood and species‐tree approaches, we recovered nearly identical topologies across datasets with 50–95% matrix completeness. We identify nine species‐level lineages in Sericomyrmex, including two new species. This is less than the previously described 19 species, even accounting for two species for which we had no UCE samples, which brings the total number of Sericomyrmex species to 11. Divergence‐dating analyses recovered 4.3 Ma as the crown‐group age estimates for Sericomyrmex, indicating a recent, rapid radiation. We also sequenced mitochondrial cytochrome oxidase subunit I (COI) for 125 specimens. Resolution and support for clades in our COI phylogeny are weak, indicating that COI is not an appropriate species‐delimitation tool. However, taxa within species consistently cluster together, suggesting that COI is useful as a species identification (‘DNA barcoding’) tool. We also sequenced internal transcribed spacer (ITS) and large subunit (LSU) for 32 Sericomyrmex fungal cultivars. The fungal phylogeny confirms that Sericomyrmex fungi are generalized higher‐attine cultivars, interspersed with Trachymyrmex‐associated fungal species, indicating cultivar sharing and horizontal transfer between these two genera. Our results indicate that UCEs offer immense potential for delimiting and resolving relationships of problematic, recently diverged species.     

Forest floor plant diversity drives the use of mature spruce forests by European bison

The distribution of large ungulates in space is in large extent driven by the availability of forage, which in temperate forests depends on light availability, and associated plant diversity and cover. We hypothesized that the increased number of GPS fixes of European bison (Bison bonasus L.) in usually avoided spruce forests was an effect of higher plant species richness and cover of the forest floor, which developed owing to increased light availability enhanced by spruce mortality. We carried out 80 forest floor plant surveys combined with tree measurement on plots chosen according to the number of GPS locations of GPS‐collared European bison. The mean plant species richness per plot was higher on intensively visited plots (IV) than rarely visited (RV) plots (30 ± 5.75 (SD) versus. 26 ± 6.19 (SD)). The frequency of 34 plant species was higher on IV plots, and they were mainly herbaceous species (32 species), while a significant part of 13 species with higher frequency on RV plots was woody plants (5 species). The species richness of forbs was higher on IV plots, while other functional groups of plants did not differ. Tree stem density on the IV plots was lower than on the RV plots (17.94 ± 6.73 (SD) versus 22.9 ± 7.67 (SD)), and the mean value of Ellenberg's ecological indicator for light availability for all forest floor plant species was higher on IV plots. European bison visiting mature spruce forests was driven by higher forest floor plant cover and species richness, and high share and species richness of forbs. The two latter features may be translated into higher quality and diversity of forage. In spite of morphological characteristics suggesting that European bison is a species of mixed (mosaic) habitats, it seems to be well adapted to thrive in diverse forests.     

Cunningham's skinks show low genetic connectivity and signatures of divergent selection across its distribution

Establishing corridors of connecting habitat has become a mainstay conservation strategy to maintain gene flow and facilitate climate‐driven range shifts. Yet, little attention has been given to ascertaining the extent to which corridors will benefit philopatric species, which might exhibit localized adaptation. Measures of genetic connectivity and adaptive genetic variation across species’ ranges can help fill this knowledge gap. Here, we characterized the spatial genetic structure of Cunningham's skink (Egernia cunninghami), a philopatric species distributed along Australia's Great Dividing Range, and assessed evidence of localized adaptation. Analysis of 4,274 SNPs from 94 individuals sampled at four localities spanning 500 km and 4° of latitude revealed strong genetic structuring at neutral loci (mean F_ST ± SD = 0.603 ± 0.237) among the localities. Putatively neutral SNPs and those under divergent selection yielded contrasting spatial patterns, with the latter identifying two genetically distinct clusters. Given low genetic connectivity of the four localities, we suggest that the natural movement rate of this species is insufficient to keep pace with spatial shifts to its climate envelope, irrespective of habitat availability. In addition, our finding of localized adaptation highlights the risk of outbreeding depression should the translocation of individuals be adopted as a conservation management strategy.     

Microsatellites for diversity studies in the golden hamster (Mesocricetus auratus)

Ten polymorphic microsatellites were developed for the golden hamster (Mesocricetus auratus), a widely used model organism in biological and medical researches. All loci were used to analyse the microsatellite variability in wild golden hamsters from Syria and in a sample of domestic animals comprising different strains. Average mean expected heterozygosity (H_E) and mean allele number (A) of domestic hamsters measured 0.279 ± 0.058 and 2.6 ± 0.306, respectively, compared to 0.809 ± 0.019 and 8.3 ± 1.075 found for wild hamsters. Cross‐species application in other Mesocricetus species proved conservation of most loci throughout the genus.     

Effective number of white shark (Carcharodon carcharias,Linnaeus) breeders is stable over four successive years in the population adjacent to eastern Australia and New Zealand

Population size is a central parameter for conservation; however, monitoring abundance is often problematic for threatened marine species. Despite substantial investment in research, many marine species remain data‐poor presenting barriers to the evaluation of conservation management outcomes and the modeling of future solutions. Such is the case for the white shark (Carcharodon carcharias), a highly mobile apex predator for whom recent and substantial population declines have been recorded in many globally distributed populations. Here, we estimate the effective number of breeders that successfully contribute offspring in one reproductive cycle (Nb) to provide a snapshot of recent reproductive effort in an east Australian–New Zealand population of white shark. Nb was estimated over four consecutive age cohorts (2010, 2011, 2012, and 2013) using two genetic estimators (linkage disequilibrium; LD and sibship assignment; SA) based on genetic data derived from two types of genetic markers (single nucleotide polymorphisms; SNPs and microsatellite loci). While estimates of Nb using different marker types produced comparable estimates, microsatellite loci were the least precise. The LD and SA estimates of Nb within cohorts using SNPs were comparable; for example, the 2013 age cohort Nb(SA) was 289 (95% CI 200–461) and Nb(LD) was 208.5 (95% CI 116.4–712.7). We show that over the time period studied, Nb was stable and ranged between 206.1 (SD ± 45.9) and 252.0 (SD ± 46.7) per year using a combined estimate of Nb(LD+SA) from SNP loci. In addition, a simulation approach showed that in this population the effective population size (Ne) per generation can be expected to be larger than Nb per reproductive cycle. This study demonstrates how breeding population size can be monitored over time to provide insight into the effectiveness of recovery and conservation measures for the white shark, where the methods described here may be applicable to other data‐poor species of conservation concern.     

Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize,Central America using cross‐species microsatellites and faecal DNA

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co‐occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross‐species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of H_E = 0.60 ± 0.18 (SD) for jaguars, H_E = 0.65 ± 0.21 (SD) for pumas and H_E = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field‐collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.     

Picking holes in traditional species delimitations: an integrative taxonomic reassessment of the Parmotrema perforatum group (Parmeliaceae,Ascomycota)

Accurate species delimitation is important as species are a fundamental unit in ecological, evolutionary and conservation biology research. In lichenized fungi, species delimitation has been difficult due to a lack of taxonomically important characteristics and due to the limits of traditional, morphology‐based species concepts. In this study we reassess the current taxonomy of the Parmotrema perforatum group, which recognizes six closely related species divided into three species pairs, each pair comprising one apotheciate (sexual) and one sorediate (asexual) species. Each pair is further characterized by a distinct combination of secondary metabolites. It was hypothesized that the three apotheciate species are reproductively isolated sibling species and that each sorediate species evolved once from the chemically identical apotheciate species. In this study, species boundaries were re‐examined using an integrative approach incorporating morphological, chemical and molecular sequence data to delimit species boundaries. Phylogenetic trees were inferred from a seven‐locus DNA sequence dataset using concatenated gene tree and coalescent‐based species‐tree inference methods. Furthermore, we employed a multi‐species coalescent method to validate candidate species. Micromorphological measurements of conidia were found to be congruent with phylogenetic clusters. Each approach that we applied to the P. perforatum group consistently recovered four of the currently circumscribed species (P. perforatum, P. hypotropum, P. subrigidum and P. louisianae), whereas P. preperforatum and P. hypoleucinum were consistently combined and are thus interpreted as conspecific.     

Isotopic niche differs between seal and fish‐eating killer whales (Orcinus orca) in northern Norway

Ecological diversity has been reported for killer whales (Orcinus orca) throughout the North Atlantic but patterns of prey specialization have remained poorly understood. We quantify interindividual dietary variations in killer whales (n = 38) sampled throughout the year in 2017–2018 in northern Norway using stable isotopic nitrogen (δ¹⁵N: ¹⁵N/¹⁴N) and carbon (δ¹³C: ¹³C/¹²C) ratios. A Gaussian mixture model assigned sampled individuals to three differentiated clusters, characterized by disparate nonoverlapping isotopic niches, that were consistent with predatory field observations: seal‐eaters, herring‐eaters, and lumpfish‐eaters. Seal‐eaters showed higher δ¹⁵N values (mean ± SD: 12.6 ± 0.3‰, range = 12.3–13.2‰, n = 10) compared to herring‐eaters (mean ± SD: 11.7 ± 0.2‰, range = 11.4–11.9‰, n = 19) and lumpfish‐eaters (mean ± SD: 11.6 ± 0.2‰, range = 11.3–11.9, n = 9). Elevated δ¹⁵N values for seal‐eaters, regardless of sampling season, confirmed feeding at high trophic levels throughout the year. However, a wide isotopic niche and low measured δ¹⁵N values in the seal‐eaters, compared to that of whales that would eat solely seals (δ_N‐measured = 12.6 vs. δ_N‐expected = 15.5), indicated a diverse diet that includes both fish and mammal prey. A narrow niche for killer whales sampled at herring and lumpfish seasonal grounds supported seasonal prey specialization reflective of local peaks in prey abundance for the two fish‐eating groups. Our results, thus, show differences in prey specialization within this killer whale population in Norway and that the episodic observations of killer whales feeding on prey other than fish are a consistent behavior, as reflected in different isotopic niches between seal and fish‐eating individuals.     

Caribbean massive corals not recovering from repeated thermal stress events during 2005–2013

Massive coral bleaching events associated with high sea surface temperatures are forecast to become more frequent and severe in the future due to climate change. Monitoring colony recovery from bleaching disturbances over multiyear time frames is important for improving predictions of future coral community changes. However, there are currently few multiyear studies describing long‐term outcomes for coral colonies following acute bleaching events. We recorded colony pigmentation and size for bleached and unbleached groups of co‐located conspecifics of three major reef‐building scleractinian corals (Orbicella franksi, Siderastrea siderea, and Stephanocoenia michelini; n = 198 total) in Bocas del Toro, Panama, during the major 2005 bleaching event and then monitored pigmentation status and changes live tissue colony size for 8 years (2005–2013). Corals that were bleached in 2005 demonstrated markedly different response trajectories compared to unbleached colony groups, with extensive live tissue loss for bleached corals of all species following bleaching, with mean live tissue losses per colony 9 months postbleaching of 26.2% (±5.4 SE) for O. franksi, 35.7% (±4.7 SE) for S. michelini, and 11.2% (±3.9 SE) for S. siderea. Two species, O. franksi and S. michelini, later recovered to net positive growth, which continued until a second thermal stress event in 2010. Following this event, all species again lost tissue, with previously unbleached colony species groups experiencing greater declines than conspecific sample groups, which were previously bleached, indicating a possible positive acclimative response. However, despite this beneficial effect for previously bleached corals, all groups experienced substantial net tissue loss between 2005 and 2013, indicating that many important Caribbean reef‐building corals will likely suffer continued tissue loss and may be unable to maintain current benthic coverage when faced with future thermal stress forecast for the region, even with potential benefits from bleaching‐related acclimation.     

Disintegrating over space and time: Paraphyly and species delimitation in the Wehrle's Salamander complex

Accurate species delimitation is critical for biodiversity studies. However, species complexes characterized by introgression, high levels of population structure and subtle phenotypic differentiation can be challenging to delimit. Here, we report on a molecular systematic investigation of the woodland salamanders Plethodon wehrlei and Plethodon punctatus, which traditionally have been placed in the Plethodon wehrlei species group. To quantify patterns of genetic variation, we collected genetic samples from throughout the range of both species, including 22 individuals from nine populations of P. punctatus, and 60 individuals from 26 populations of P. wehrlei. From these samples, we sequenced three mtDNA loci (5596 base pairs) and five nuclear loci (3377 base pairs). We inferred time‐calibrated gene trees and species trees using BEAST 2.4.6, and we delimited putative species using a Bayesian implementation of the general mixed Yule‐coalescent model (bGMYC) and STRUCTURE. Finally, we validated putative species using the multispecies coalescent as implemented in Bayesian Phylogenetics and Phylogeography (BPP). We found substantial phylogeographic diversity in P. wehrlei, including multiple geographically cohesive clades and an inferred mitochondrial common ancestor at 11.5 myr (95% HPD: 9.6–13.6 myr) that separated populations formerly assigned to P. dixi from all other populations. We also found that P. punctatus is deeply nested within P. wehrlei, rendering the latter paraphyletic. After discussing the challenges faced by modern species delimitation methods, we recommend retaining P. punctatus because it is ecologically and phenotypically distinct. We further recommend that P. dixi be recognized as a valid species.     

One species hides many: Molecular and morphological evidence for cryptic speciation in a thread snake (Leptotyphlopidae: Leptotyphlops sylvicolus Broadley & Wallach, 1997)

We investigate the phylogeographic structure of a fossorial forest‐living snake species, the forest thread snake, Leptotyphlopssylvicolus Broadley & Wallach, 1997 by sampling specimens from the Eastern Cape and KwaZulu‐Natal provinces of South Africa. Phylogenetic results, using Bayesian inferences and maximum likelihood, from the combined mitochondrial sequence data (cyt b and ND4), along with population genetic analyses suggest the presence of phylogeographic breaks broadly congruent to those exhibited by other forest‐living taxa. Divergence‐time estimates indicate that cladogenesis within the study taxon occurred during the late Miocene climatic shifts, suggesting that cladogenesis was driven by habitat fragmentation. We further investigate the species‐level divergence within L. sylvicolus by including two partial nuclear loci (PRLR and RAG1). The three species delimitation methods (ABGD, bGMYC, and STACEY), retrieved 10–12 putative species nested within the L. sylvicolus species complex. These results were corroborated by iBPP implementing molecular and morphological data in an integrative Bayesian framework. The morphological analyses exhibit large overlap among putative species but indicate differences between grassland and forest species. Due to the narrow distributions of these putative species, the results of the present study have further implications for the conservation status of the L. sylvicolus species complex and suggest that forest and grassland habitats along the east coast of South Africa may harbor significantly higher levels of diversity than currently recognized.     

Length–weight relationship of six deep‐sea fish species from the shelf regions of western Bay of Bengal and Andaman waters

Length–weight relationships for six deep‐sea fish species inhabiting the shelf regions of the western Bay of Bengal and the waters of Andaman and Nicobar of India are presented. Samples were collected using high‐speed demersal trawl and expo demersal trawl nets at depths of 231–514 m in August and September 2010. The b values in the analyses ranged from 3.05 to 3.31, showing a mean and median value of 3.21 (SE ± 0.039, SD ± 0.097) and 3.2, respectively. Comparisons of b values with earlier estimations confirm the presence of spatial and temporal variations in the length–weight relations among the species. Coefficient of determination scores ranged from 0.94 to 0.97, indicating robustness of the samples analysed. This study provides the first estimates of length–weight relationships for four of the deep‐sea fishes, enriching the understanding of the growth patterns and population dynamics of these less‐studied deep‐sea fishery resources in Indian Ocean waters.     

Discovery of a multispecies shark aggregation and parturition area in the Ba Estuary,Fiji Islands

Population declines in shark species have been reported on local and global scales, with overfishing, habitat destruction and climate change posing severe threats. The lack of species‐specific baseline data on ecology and distribution of many sharks, however, makes conservation measures challenging. Here, we present a fisheries‐independent shark survey from the Fiji Islands, where scientific knowledge on locally occurring elasmobranchs is largely still lacking despite the location's role as a shark hotspot in the Pacific. Juvenile shark abundance in the fishing grounds of the Ba Estuary (north‐western Viti Levu) was assessed with a gillnet‐ and longline‐based survey from December 2015 to April 2016. A total of 103 juvenile sharks identified as blacktip Carcharhinus limbatus (n = 57), scalloped hammerhead Sphyrna lewini (n = 35), and great hammerhead Sphyrna mokarran (n = 11) sharks were captured, tagged, and released. The condition of umbilical scars (68% open or semihealed), mean sizes of individuals (±SD) (C. limbatus: 66.5 ± 3.8 cm, S. lewini: 51.8 ± 4.8 cm, S. mokarran 77.4 ± 2.8 cm), and the presence of these species over recent years (based on fishermen interviews), suggest that the Ba Estuary area is a critical habitat for multiple species that are classified as “Near Threatened” or “Endangered.” Specifically, the area likely acts as a parturition ground over the studied period, and potentially as a subsequent nursery area. We identified subareas of high abundance and found that temperature, salinity and depth acted as small‐scale environmental drivers of shark abundance. The data suggests a tendency for species‐specific spatial use, both horizontally (i.e., between sampling areas) and vertically (i.e., across the water column). These results enhance the understanding of shark ecology in Fiji and provide a scientific basis for the implementation of local conservation strategies that contribute to the protection of these threatened species.     

Estimating seed dispersal distance: A comparison of methods using animal movement and plant genetic data on two primate‐dispersed Neotropical plant species

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Morphotypes or distinct species? A multilocus assessment of two East Asian scimitar babblers (Aves,Timaliidae)

In birds, widespread species complexes often exhibit dramatic plumage differences across their distribution, which can give rise to discordance between morphotypes and phylogroups. Accurate identification of species diversity may require an integrated approach in which multilocus genetic data are used for inference and further corroborated by ecological, morphometric or behavioural data. Pomatorhinus gravivox and P. swinhoei, which were formerly considered component races of P. erythrogenys, were recently split using a delimitation system that mainly relied on the quantification of differences in phenotypic traits. We therefore carried out a reassessment of this taxonomic recommendation by conducting phylogenetic and population genetic analyses with multilocus genetic data. A deep mitochondrial split with a Kimura 2-parameter distance of 0.061 was observed, mainly corresponding to the two morphologically defined species. However, one individual from P. swinhoei harboured a haplotype of P. gravivox. Individual-based analyses of nuclear loci identified two distinct clusters that were exactly congruent with the two species. BPP delimitation also provided support for the separation. These congruencies support the notion that these two taxa are best regarded as two separate species. The presence of the P. gravivox mitochondrial haplotype in P. swinhoei was most likely a result of hybridization, due to the clear separation of nuclear loci. The speciation might be attributed to paleoclimatic changes but requires further evaluation due to the likelihood of ecological speciation. This study was in accordance with the results inferred from the quantitative system but highlighted the importance of sampling various data, especially in contact zones, in the study of taxonomy and speciation history.