Discordance between morphological and molecular species boundaries among Caribbean species of the reef sponge Callyspongia

Sponges are among the most species‐rich and ecologically important taxa on coral reefs, yet documenting their diversity is difficult due to the simplicity and plasticity of their morphological characters. Genetic attempts to identify species are hampered by the slow rate of mitochondrial sequence evolution characteristic of sponges and some other basal metazoans. Here we determine species boundaries of the Caribbean coral reef sponge genus Callyspongia using a multilocus, model‐based approach. Based on sequence data from one mitochondrial (COI), one ribosomal (28S), and two single‐copy nuclear protein‐coding genes, we found evolutionarily distinct lineages were not concordant with current species designations in Callyspongia. While C. fallax, C. tenerrima, and C. plicifera were reciprocally monophyletic, four taxa with different morphologies (C. armigera, C. longissima, C. eschrichtii, and C. vaginalis) formed a monophyletic group and genetic distances among these taxa overlapped distances within them. A model‐based method of species delimitation supported collapsing these four into a single evolutionary lineage. Variation in spicule size among these four taxa was partitioned geographically, not by current species designations, indicating that in Callyspongia, these key taxonomic characters are poor indicators of genetic differentiation. Taken together, our results suggest a complex relationship between morphology and species boundaries in sponges.     

The power and perils of 'molecular taxonomy': a case study of eyeless and endangered Cicurina (Araneae: Dictynidae) from Texas caves

Rapid development in karst-rich regions of the US state of Texas has prompted the listing of four Cicurina species (Araneae, Dictynidae) as US Federally Endangered. A major constraint in the management of these taxa is the extreme rarity of adult specimens, which are required for accurate species identification. We report a first attempt at using mitochondrial DNA (mtDNA) sequences to accurately identify immature Cicurina specimens. This identification is founded on a phylogenetic framework that is anchored by identified adult and/or topotypic specimens. Analysis of approximately 1 kb of cytochrome oxidase subunit I (CO1) mtDNA data for over 100 samples results in a phylogenetic tree that includes a large number of distinctive, easily recognizable, tip clades. These tip clades almost always correspond to a priori species hypotheses, and show nonoverlapping patterns of sequence divergence, making it possible to place species names on a number of immature specimens. Three cases of inconsistency between recovered tip clades and a priori species hypotheses suggest possible introgression between cave-dwelling Cicurina, or alternatively, species synonymy. Although species determination is not possible in these instances, the inconsistencies point to areas of taxonomic ambiguity that require further study. Our molecular phylogenetic sample is largest for the Federally Endangered C. madla. These data suggest that C. madla occurs in more than twice the number of caves as previously reported, and indicate the possible synonymy of C. madla with C. vespera, which is also Federally Endangered. Network analyses reveal considerable genetic divergence and structuring across caves in this species. Although the use of DNA sequences to identify previously 'unidentifiable' specimens illustrates the potential power of molecular data in taxonomy, many other aspects of the same dataset speak to the necessity of a balanced taxonomic approach.     

Species delimitation in Neotropical fishes of the genus Characidium (Teleostei,Characiformes)

Neotropical darters of the genus Characidium have a complex systematic history with several examples of sympatry throughout their distribution range in Neotropical freshwaters. Although various species within the genus have been used as models to investigate chromosomal evolution and biogeography, species boundaries and relationships still remain uncertain. Here, we use mitochondrial DNA sequences to perform species delimitation analyses within Characidium and test previous hypotheses of species richness within the Characidium zebra complex and among sympatric morphotypes of C. alipioi. Results indicate high genetic distances within tested species complexes and revealed the presence of strongly supported lineages such as the large C. lauroi group from southeastern Brazil. This suggests that the evolutionary history of these groups may be correlated with biogeographic history. Analyses also reveal that three geographically isolated populations of C. zebra represent a single species, leading us to reject prior hypothesis of multiple species. Species delimitation using mitochondrial data strongly supports the presence of two sympatric species within C. alipioi in southeastern Brazil despite limited morphological variation and conserved chromosomal patterns. These results provide a framework to further the study of systematics and evolution within Characidium.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Genetic evidence challenges the native status of a threatened freshwater fish (Carassius carassius) in England

A fundamental consideration for the conservation of a species is the extent of its native range, that is, regions naturally colonized. However, both natural processes and human‐mediated introductions can drive species distribution shifts. Ruling out the human‐mediated introduction of a species into a given region is vital for its conservation, but remains a significant challenge in most cases. The crucian carp Carassius carassius (L.) is a threatened freshwater fish thought to be native to much of Europe. However, its native status in England is based only on anecdotal evidence. Here, we devise an approach that can be used to empirically test the native status of English fauna. We use this approach, along with 13 microsatellite loci, population structure analyses, and Approximate Bayesian Computation (ABC), to test hypotheses for the origins of C. carassius in England. Contrary to the current consensus, we find strong support for the human‐mediated introduction of C. carassius into England during the 15th century. This result stimulates an interesting and timely debate surrounding motivations for the conservation of species. We discuss this topic, and the potential for continued conservation of C. carassius in England, despite its non‐native origins.     

Resolving species boundaries in the critically imperiled freshwater mussel species,Fusconaia mitchelli (Bivalvia: Unionidae)

Species are a fundamental unit of biology, and defining accurate species boundaries is integral to effective conservation and management of imperiled taxa. Freshwater mussels (Bivalvia: Unionidae) are among the most imperiled groups of organisms in North America, yet species boundaries remain uncertain for many taxa. The False Spike, Fusconaia mitchelli (Simpson in Dall, 1895), is a freshwater mussel considered to be endemic to central Texas (Brazos, Colorado, and Guadalupe drainages). Recent research revealed significant intraspecific genetic variation between geographically separated populations of F. mitchelli, which could be indicative of speciation; however, small sample sizes for several of the populations precluded formal taxonomic revision. Here, we increase taxon sampling and use multilocus DNA sequence data and traditional morphometrics to re‐evaluate species boundaries in F. mitchelli. We sequenced three loci: the protein‐coding mitochondrial DNA genes cytochrome c oxidase subunit 1 and NADH dehydrogenase 1, and the nuclear internal transcribed spacer 1. Phylogenetic analyses depicted deep genetic divergence between F. mitchelli in the Guadalupe and those in the Brazos and Colorado drainages, which was further supported by available biogeographic information. Morphometric analyses and coalescent‐based species delimitation models integrating both DNA sequence and morphological data provided strong support for the divergence observed between the two geographically isolated clades of F. mitchelli. Based on these results, we revise taxonomy accordingly by elevating the junior synonym Fusconaia iheringi (Wright, 1898) to represent the Brazos and Colorado populations and restrict the distribution of F. mitchelli to the Guadalupe River drainage. Our findings may impact pending management decisions to protect F. mitchelli under the U.S. Endangered Species Act.     

New evidence of unexpectedly high animal density and diet diversity will benefit the conservation of the Critically Endangered western ringtail possum

A comprehensive and contemporary understanding of habitat and resource requirements has been critical to the conservation of multiple taxa and ecosystems globally. Until recently, much of the ecological knowledge that contributes to conservation priorities and strategies for the Critically Endangered western ringtail possum (Pseudocheirus occidentalis) was largely derived from decades‐old observations in peppermint (Agonis flexuosa) and marri‐jarrah (Corymbia calophylla and Eucalyptus marginanta) woodlands in the northern parts of the species range. These observations do not account for more recent evidence of their flexible use of habitat resources in other regions of its range. This may represent a significant conservation opportunity for the species through the identification of additional habitats that warrant protection. In a region where knowledge of their ecology is scarce, we used scat analysis and quantitative spotlighting to determine the diet and density of western ringtail possums in three vegetation types: peppermint, sheoak (Allocasuarina fraseriana) and marri‐eucalypt (C. calophylla, E. marginanta and Eucalyptus staerii) woodlands. Given the species’ reported dependence on peppermint woodlands and dominant canopy species for food sources, we hypothesised that western ringtail possums would be most abundant in this habitat type and that their diet would comprise the foliage of few (≤2 species) canopy species. We found western ringtail possums consumed a higher diversity of plant species than expected (8–14), exhibited dietary preference for non‐dominant canopy species and were present in all sampled vegetation types at substantially higher densities than previously recorded for the region (as high as 17 possums ha^?1). Our results confirm (i) the western ringtail possum is flexible in its use of habitat resources and (ii) the significant conservation value of sheoak and marri‐eucalypt woodlands in the southernmost portion of its distribution.     

Species delimitation and phylogeography of Abies delavayi complex: Inferred from morphological,molecular, and climatic data

The species delimitation within Abies delavayi complex, consisting of A. delavayi Franch., A. fabri (Mast.) Craib, A. nukiangensis W. C. Cheng & L. K. Fu, and A. delavayi subsp. fansipanensis (Q. P. Xiang, L. K. Fu & Nan Li) Rushforth, has been a subject of long‐term dispute. We combined different lines of evidence, including morphological characters, population‐based mitochondrial DNA and chloroplast DNA sequences, and plastome data to assess its species delimitation. These four independent datasets revealed a consistent result that the four taxa of A. delavayi complex were hardly separated and could be regarded as one species. Our results further suggested that these four taxa might have experienced rapid morphological diversification, following a common expansion triggered by climate oscillations during the Pleistocene. In addition, we surveyed the phylogeographical history of A. delavayi complex as a whole based on ecological niche models and molecular data. These results consistently revealed that this high‐mountain fir complex had experienced glacial expansion and interglacial contraction. Therefore, we propose that A. delavayi complex probably has undergone the phalanx expansion model in response to climate change during the Quaternary. Such expansion patterns demand consideration when establishing conservation strategies for threatened fir species.     

Parnassia zhengyuana sp. nov. and P. simianshanensis sp. nov.: two new species of Parnassia (Celastraceae) from karst caves and Danxia landform in southwest China

Two new species of Parnassia, Parnassia zhengyuana M.X. Ren & J. Zhang and P. simianshanensis M.X. Ren, J. Zhang & Z.Y. Liu, have been found in mountainous areas of Chongqing City, southwest China, and are described and illustrated here. Parnassia zhengyuana is found in a limestone cave near Mt Jinfo, Nanchuan District, Chongqing City. This tiny plant has special spoon‐shaped leaves and undivided staminodes with green apices. Parnassia simianshanensis was discovered in Mt Simian, Jiangjin County, Chongqing City. This species grows along streams on red sandstones, i.e. in Danxia landform. It can be easily identified by its unique rhombic leaves and the five‐ or four‐branched staminodes. The conservation status of P. zhengyuana and P. simianshanensis are assessed respectively as ‘Critically Endangered’ (CR) and ‘VU’ (Vulnerable) according to IUCN criteria. The roles of karst and Danxia landforms in the speciation process are discussed.     

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.     

Cutting the Gordian Knot: Phylogenetic and ecological diversification of the Mesalina brevirostris species complex (Squamata,Lacertidae)

Mesalina are small lacertid lizards occurring in the Saharo‐Sindian deserts from North Africa to the east of the Iranian plateau. Earlier phylogenetic studies indicated that there are several species complexes within the genus and that thorough taxonomic revisions are needed. In this study, we aim at resolving the phylogeny and taxonomy of the M. brevirostris species complex distributed from the Middle East to the Arabian/Persian Gulf region and Pakistan. We sequenced three mitochondrial and three nuclear gene fragments, and in combination with species delimitation and species‐tree estimation, we infer a time‐calibrated phylogeny of the complex. The results of the genetic analyses support the presence of four clearly delimited species in the complex that diverged approximately between the middle Pliocene and the Pliocene/Pleistocene boundary. Species distribution models of the four species show that the areas of suitable habitat are geographically well delineated and nearly allopatric, and that most of the species have rather divergent environmental niches. Morphological characters also confirm the differences between the species, although sometimes minute. As a result of all these lines of evidence, we revise the taxonomy of the Mesalina brevirostris species complex. We designate a lectotype for Mesalina brevirostris Blanford, 1874; resurrect the available name Eremias bernoullii Schenkel, 1901 from the synonymy of M. brevirostris; elevate M. brevirostris microlepis (Angel, 1936) to species status; and describe Mesalina saudiarabica, a new species from Saudi Arabia.     

Evolution of the African slippery frogs (Anura: Conraua), including the world’s largest living frog

Although Conraua goliath is well known as the largest living frog species, the diversity and evolution of the genus Conraua across sub-Saharan Africa remain poorly understood. We present multilocus phylogenetic analyses of the six currently recognized species that provide insights into divergence times, biogeography, body size evolution and undescribed species. An analysis of divergence times demonstrates that crown-group Conraua arose some time during the latest Oligocene to mid-Miocene followed by divergence into major lineages in the mid-Miocene that may reflect the fragmentation of widespread tropical forests in Africa that began at this time. We find three pairs of sister species, C. crassipes + C. beccarii, C. alleni + C. derooi and C. goliath + C. robusta, each of which diverged during the Miocene. These relationships reject phylogenetic hypotheses based solely on biogeography as the geographically peripheral C. beccarii from north-eastern Africa is nested within western African species and the Central African species do not form a clade. Our species delimitation analyses provide support for undescribed species in C. alleni, C. beccarii and C. derooi, and possibly C. crassipes, suggesting that the current taxonomy substantially underestimates species diversity. There is no clear directional trend of either increasing or decreasing body size in Conraua and the three largest species do not form a clade. With a robust phylogenetic hypothesis in hand, further field-based studies are needed to understand the evolution of morphology and life history in this charismatic African anuran clade.     

Protecting a single endangered species and meeting multiple conservation goals: an approach with Guaiacum sanctum in Yucatan Peninsula,Mexico

Aim New protected areas should consider safeguarding high conservation value sites based on multiple criteria and not just the presence of a single endangered or charismatic species. However, the extent to which complementary criteria coincide is usually unknown. We use the case of Guaiacum sanctum (Zygopyllaceae), an endangered timber tree species, to explore whether the protection of forests where this species is most abundant would meet other complementary conservation goals, such as capturing regional plant biodiversity, protecting other threatened/endemic species or safeguarding ecosystem services. Location Yucatan Peninsula, southern Mexico. Methods We conducted an analysis of the structure, composition and diversity of tree communities (including stems ≥5 cm dbh) at eight G. sanctum forest sites. We identified endemic and threatened tree species and quantified above‐ground tree biomass and carbon storage in these G. sanctum forests. Results Guaiacum sanctum forests contain 35–59 tree species on plots as small as 1000 m². The species composition of tree communities changed rapidly (high β‐diversity) across soil boundaries and rainfall regimes. Twenty‐one endemic and eight threatened tree species were recorded in our inventories. Individuals of G. sanctum represented up to 55% of the above‐ground carbon for trees ≥5 cm dbh. The high basal area of G. sanctum forests plus the high wood density, abundance, large size and longevity (more than 500 years) of G. sanctum and other tree species enhance the potential importance of these forests for carbon storage. Main conclusions A conservation strategy focused on protecting important populations of G. sanctum in the Yucatan Peninsula would have significant co‐benefits for conservation of regional tree species biodiversity and provision of critical ecosystem services. Our study illustrates a multiple criteria approach useful for the selection of areas with high conservation value on the basis of endemic, threatened species, species richness and ecosystem services.     

Vulnerable island carnivores: the endangered endemic dwarf procyonids from Cozumel Island

Insular carnivores represent some of the most critically threatened species, but also the least known. To evaluate the conservation status of these species, thorough abundance estimates are urgently required. To better understand the population biology and conservation status of the endemic and threatened pygmy raccoon (Procyon pygmaeus) and dwarf coati (Nasua nelsoni) on Cozumel Island, Mexico, we worked island-wide to identify the presence of these species, and for the pygmy raccoon we studied several populations in depth during 2001–2003. On Cozumel, trapping was conducted for >6,600 trap nights in 19 locations of varying habitat types. A total of 96 pygmy raccoons (47 males and 49 females) and a single adult, male dwarf coati (N. nelsoni) were captured. Estimated total annual pygmy raccoon population size ± SE was 80 ± 26.1, resulting in an average density of 22 ± 5.1 raccoons/km² for the three small sites where the animals persist. Based in part on the findings of this study that indicate these species have a restricted range and small population numbers, the IUCN recently changed the listing of the pygmy raccoon to Critically Endangered from Endangered. In contrast, the status of the dwarf coati (Endangered) has not been changed, although the taxon is in eminent danger of extinction and in need of immediate conservation action.     

Molecular species delimitation methods recover most song‐delimited cicada species in the European Cicadetta montana complex

Molecular species delimitation is increasingly being used to discover and illuminate species level diversity, and a number of methods have been developed. Here, we compare the ability of two molecular species delimitation methods to recover song‐delimited species in the Cicadetta montana cryptic species complex throughout Europe. Recent bioacoustics studies of male calling songs (premating reproductive barriers) have revealed cryptic species diversity in this complex. Maximum likelihood and Bayesian phylogenetic analyses were used to analyse the mitochondrial genes COI and COII and the nuclear genes EF1α and period for thirteen European Cicadetta species as well as the closely related monotypic genus Euboeana. Two molecular species delimitation methods, general mixed Yule‐coalescent (GMYC) and Bayesian phylogenetics and phylogeography, identified the majority of song‐delimited species and were largely congruent with each other. None of the molecular delimitation methods were able to fully recover a recent radiation of four Greek species.     

Speciation,population structure,and demographic history of the Mojave Fringe‐toed Lizard (Uma scoparia), a species of conservation concern

The North American deserts were impacted by both Neogene plate tectonics and Quaternary climatic fluctuations, yet it remains unclear how these events influenced speciation in this region. We tested published hypotheses regarding the timing and mode of speciation, population structure, and demographic history of the Mojave Fringe‐toed Lizard (Uma scoparia), a sand dune specialist endemic to the Mojave Desert of California and Arizona. We sampled 109 individual lizards representing 22 insular dune localities, obtained DNA sequences for 14 nuclear loci, and found that U. scoparia has low genetic diversity relative to the U. notata species complex, comparable to that of chimpanzees and southern elephant seals. Analyses of genotypes using Bayesian clustering algorithms did not identify discrete populations within U. scoparia. Using isolation‐with‐migration (IM) models and a novel coalescent‐based hypothesis testing approach, we estimated that U. scoparia diverged from U. notata in the Pleistocene epoch. The likelihood ratio test and the Akaike Information Criterion consistently rejected nested speciation models that included parameters for migration and population growth of U. scoparia. We reject the Neogene vicariance hypothesis for the speciation of U. scoparia and define this species as a single evolutionarily significant unit for conservation purposes.     

Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult

Using multiple, independent approaches to molecular species delimitation is advocated to accommodate limitations and assumptions of a single approach. Incongruence in delimitation schemes is a potential by‐product of employing multiple methods on the same data, and little attention has been paid to its reconciliation. Instead, a particular scheme is prioritized, and/or molecular delimitations are coupled with additional, independent lines of evidence that mitigate incongruence. We advocate that incongruence within a line of evidence should be accounted for before comparing across lines of evidence that can themselves be incongruent. Additionally, it is not uncommon for empiricists working in nonmodel systems to be data‐limited, generating some concern for the adequacy of available data to address the question of interest. With conservation and management decisions often hinging on the status of species, it seems prudent to understand the capabilities of approaches we use given the data we have. Here, we apply two molecular species delimitation approaches, spedeSTEM and BPP, to the Castilleja ambigua (Orobanchaceae) species complex, a relatively young plant lineage in western North America. Upon finding incongruence in our delimitation, we employed a post hoc simulation study to examine the power of these approaches to delimit species. Given the data we collected, we find that spedeSTEM lacks the power to delimit while BPP is capable, thus allowing us to address incongruence before proceeding in delimitation. We suggest post hoc simulation studies like this compliment empirical delimitation and serve as a means of exploring conflict within a line of evidence and dealing with it appropriately.     

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with morphology

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species‐specific in spiders – exhibits considerable intraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu & Kuntner, 2015, endemic to south‐east China, where males are only available for two of the seven morphological species (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI) for 51 newly collected specimens of six morphological species and analysed them using five species‐delimitation methods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD), generalized mixed Yule‐coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, completes the revision of the genus. Although multi‐locus phylogenetic approaches may be needed for complex taxonomic delimitations, our results indicate that even single‐locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation. © 2015 The Linnean Society of London