Species delimitation with limited sampling: An example from rare trapdoor spider genus Cyclocosmia (Mygalomorphae, Halonoproctidae).

The outcome of species delimitation depends on many factors, including conceptual framework, study design, data availability, methodology employed and subjective decision making. Obtaining sufficient taxon sampling in endangered or rare taxa might be difficult, particularly when non-lethal tissue collection cannot be utilized. The need to avoid overexploitation of the natural populations may thus limit methodological framework available for downstream data analyses and bias the results. We test species boundaries in rare North American trapdoor spider genus Cyclocosmia Ausserer (1871) inhabiting the Southern Coastal Plain biodiversity hotspot with the use of genomic data and two multispecies coalescent model methods. We evaluate the performance of each methodology within a limited sampling framework. To mitigate the risk of species over splitting, common in taxa with highly structured populations, we subsequently implement a species validation step via genealogical diversification index (gdi), which accounts for both genetic isolation and gene flow. We delimited eight geographically restricted lineages within sampled North American Cyclocosmia, suggesting that major river drainages in the region are likely barriers to dispersal. Our results suggest that utilizing BPP in the species discovery step might be a good option for datasets comprising hundreds of loci, but fewer individuals, which may be a common scenario for rare taxa. However, we also show that such results should be validated via gdi, in order to avoid over splitting.

Species boundaries in the messy middle-A genome-scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi.

Species delimitation among closely related species is challenging because traditional phenotype-based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high-throughput sequencing, it has become increasingly cost-effective to acquire genome-scale data which can resolve previously ambiguous species boundaries. As the availability of genome-scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro-endemism in a lineage of lichen-forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single-locus and genome-scale data consisting of (a) single-locus species delimitation analysis using ASAP, (b) maximum likelihood-based phylogenetic tree inference, (c) genome-scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross-validate results between genome-scale and single-locus datasets, differently sampled subsets of genomic data and to control for population-level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro-endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.

Rivers of Indochina as potential drivers of lineage diversification in the spotted flying lizard (Draco maculatus) species complex.

Southeast Asia hosts a rich concentration of biodiversity within multiple biodiversity hotspots. Indochina, a region with remarkably high levels of in situ diversification, possesses five major rivers (Ayeyarwady, Chiang Mai, Mekong, Red, and Salween), several of which coincide with phylogenetic breaks of terrestrial taxa. Draco maculatus possesses a range that stretches across Indochina, which widespread geographic distribution along with potential discrete variation within subspecies alludes to the possibility of this taxon constituting multiple divergent lineages. Using sequence data from three mitochondrial (12S, 16S, and ND2) and three nuclear (BDNF, CMOS, and PNN) genes, we provide the first estimated phylogeny of this hypothesized species complex and examine its phylogeographic architecture with maximum likelihood and Bayes factor delimitation (BFD) approaches. Our results support multiple divergent lineages with phylogenetic breaks coincident with rivers, indicating that river barriers may be contributing to the elevated levels of in situ diversification of Indochina.

Genomics overrules mitochondrial DNA, siding with morphology on a controversial case of species delimitation.

Species delimitation is a major quest in biology and is essential for adequate management of the organismal diversity. A challenging example comprises the fish species of red snappers in the Western Atlantic. Red snappers have been traditionally recognized as two separate species based on morphology: Lutjanus campechanus (northern red snapper) and L. purpureus (southern red snapper). Recent genetic studies using mitochondrial markers, however, failed to delineate these nominal species, leading to the current lumping of the northern and southern populations into a single species ( L. campechanus). This decision carries broad implications for conservation and management as red snappers have been commercially over-exploited across the Western Atlantic and are currently listed as vulnerable. To address this conflict, we examine genome-wide data collected throughout the range of the two species. Population genomics, phylogenetic and coalescent analyses favour the existence of two independent evolutionary lineages, a result that confirms the morphology-based delimitation scenario in agreement with conventional taxonomy. Despite finding evidence of introgression in geographically neighbouring populations in northern South America, our genomic analyses strongly support isolation and differentiation of these species, suggesting that the northern and southern red snappers should be treated as distinct taxonomic entities.

Genome-wide SNP data and morphology support the distinction of two new species of Kovarikia Soleglad, Fet & Graham, 2014 endemic to California (Scorpiones, Vaejovidae).

Morphologically conserved taxa such as scorpions represent a challenge to delimit. We recently discovered populations of scorpions in the genus Kovarikia Soleglad, Fet & Graham, 2014 on two isolated mountain ranges in southern California. We generated genome-wide single nucleotide polymorphism data and used Bayes factors species delimitation to compare alternative species delimitation scenarios which variously placed scorpions from the two localities with geographically adjacent species or into separate lineages. We also estimated a time-calibrated phylogeny of Kovarikia and examined and compared the morphology of preserved specimens from across its distribution. Genetic results strongly support the distinction of two new lineages, which we describe and name here. Morphology among the species of Kovarikia was relatively conserved, despite deep genetic divergences, consistent with recent studies of stenotopic scorpions with limited vagility. Phylogeographic structure discovered in several previously described species also suggests additional cryptic species are probably present in the genus.

Cryptic diversity, pathogenicity, and evolutionary species boundaries in Cercospora populations associated with Cercospora leaf spot of Beta vulgaris.

The taxonomy and evolutionary species boundaries in a global collection of Cercospora isolates from Beta vulgaris was investigated based on sequences of six loci. Species boundaries were assessed using concatenated multi-locus phylogenies, Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Processes (PTP), and Bayes factor delimitation (BFD) framework. Cercospora beticola was confirmed as the primary cause of Cercospora leaf spot (CLS) on B. vulgaris. Cercospora apii, C. cf. flagellaris, Cercospora sp. G, and C. zebrina were also identified in association with CLS on B. vulgaris. Cercospora apii and C. cf. flagellaris were pathogenic to table beet but Cercospora sp. G and C. zebrina did not cause disease. Genealogical concordance phylogenetic species recognition, GMYC and PTP methods failed to differentiate C. apii and C. beticola as separate species. On the other hand, multi-species coalescent analysis based on BFD supported separation of C. apii and C. beticola into distinct species; and provided evidence of evolutionary independent lineages within C. beticola. Extensive intra- and intergenic recombination, incomplete lineage sorting and dominance of clonal reproduction complicate evolutionary species recognition in the genus Cercospora. The results warrant morphological and phylogenetic studies to disentangle cryptic speciation within C. beticola.

"Fix Me Another Marguerite!": Species delimitation in a group of intensively hybridizing lineages of ox-eye daisies (Leucanthemum Mill., Compositae-Anthemideae).

Delineating species boundaries in the framework of the multi-species coalescent (MSC) proves to be a reliable, objective, and reproducible method in an increasing number of studies. However, the underlying model assumes the lack of gene flow after speciation; an assumption which may be frequently violated in plant evolution. This study evaluates the robustness of currently available species delimitation methods implemented in beast (BFD, BFD*, and dissect) in the closely-knit ox-eye daisy group around Leucanthemum ageratifolium Pau. Comprising five taxa being allopatrically distributed between northern Spain and southern Italy this study group shows signs of hybridization with the widespread and codistributed species Leucanthemum vulgare (Vaill.) Lam. to various extent. As expected, our empirical analyses based on both AFLP fingerprinting and sequence data demonstrate that the robustness of species delimitation results is considerably influenced by the intensity of hybridization among species and the number of hybrid individuals included. Therefore, we set up a methodological pipeline with a first step of identification and subsequent removal of individuals showing admixed genetic patterns caused by actual interbreeding using AFLP-fingerprint and morphometric data, followed by application of different Bayesian MSC species delimitation methods based on the remnant individuals using both AFLP-fingerprint and sequence data (four nuclear markers, five concatenated intergenic spacer regions of the plastid genome). The results argue for acknowledgement of Leucanthemum laciniatum, L. legraeanum, and L. ligusticum as independent species, show the close relationship of L. ageratifolium, L. monspeliense, and L. vulgare, and give rise to the description of three nothospecies new to science.

New species in the Sitalcina sura species group (Opiliones, Laniatores, Phalangodidae), with evidence for a biogeographic link between California desert canyons and Arizona sky islands.

The western United States is home to numerous narrowly endemic harvestman taxa (Arachnida, Opiliones), including members of the genus Sitalcina Banks, 1911. Sitalcina is comprised of three species groups, including the monospecific Sitalcina californica and Sitalcina lobata groups, and the Sitalcina sura group with eight described species. All species in the Sitalcina sura group have very small geographic distributions, with group members distributed like disjunct "beads on a string" from Monterey south to southern California and southeast to the sky-island mountain ranges of southern Arizona. Here, molecular phylogenetic and species delimitation analyses were conducted for all described species in the Sitalcina sura group, plus several newly discovered populations. Species trees were reconstructed using multispecies coalescent methods implemented in *BEAST, and species delimitation was accomplished using Bayes Factor Delimitation (BFD). Based on quantitative species delimitation results supported by consideration of morphological characters, two new species (Sitalcina oasiensis sp. n., Sitalcina ubicki sp. n.) are described. We also provide a description of the previously unknown male of Sitalcina borregoensis Briggs, 1968. Molecular phylogenetic evidence strongly supports distinctive desert versus coastal clades, with desert canyon taxa from southern California more closely related to Arizona taxa than to geographically proximate California coastal taxa. We hypothesize that southern ancestry and plate tectonics have played a role in the diversification history of this animal lineage, similar to sclerophyllous plant taxa of the Madro-Tertiary Geoflora. Molecular clock analyses for the Sitalcina sura group are generally consistent with these hypotheses. We also propose that additional Sitalcina species await discovery in the desert canyons of southern California and northern Baja, and the mountains of northwestern mainland Mexico.

A new monster from southwest Oregon forests: Cryptomaster behemoth sp. n. (Opiliones, Laniatores, Travunioidea).

The monotypic genus Cryptomaster Briggs, 1969 was described based on individuals from a single locality in southwestern Oregon. The described species Cryptomaster leviathan Briggs, 1969 was named for its large body size compared to most travunioid Laniatores. However, as the generic name suggests, Cryptomaster are notoriously difficult to find, and few subsequent collections have been recorded for this genus. Here, we increase sampling of Cryptomaster to 15 localities, extending their known range from the Coast Range northeast to the western Cascade Mountains of southern Oregon. Phylogenetic analyses of mitochondrial and nuclear DNA sequence data reveal deep phylogenetic breaks consistent with independently evolving lineages. We use discovery and validation species delimitation approaches to generate and test species hypotheses, including a coalescent species delimitation method to test multi-species hypotheses. For delimited species, we use light microscopy and SEM to discover diagnostic morphological characters. Although Cryptomaster has a small geographic distribution, this taxon is consistent with other short-range endemics in having deep phylogenetic breaks indicative of species level divergences. Herein we describe Cryptomaster behemoth sp. n., and provide morphological diagnostic characters for identifying Cryptomaster leviathan and Cryptomaster behemoth.

Sky island diversification meets the multispecies coalescent - divergence in the spruce-fir moss spider (Microhexura montivaga, Araneae, Mygalomorphae) on the highest peaks of southern Appalachia.

Microhexura montivaga is a miniature tarantula-like spider endemic to the highest peaks of the southern Appalachian mountains and is known only from six allopatric, highly disjunct montane populations. Because of severe declines in spruce-fir forest in the late 20th century, M. montivaga was formally listed as a US federally endangered species in 1995. Using DNA sequence data from one mitochondrial and seven nuclear genes, patterns of multigenic genetic divergence were assessed for six montane populations. Independent mitochondrial and nuclear discovery analyses reveal obvious genetic fragmentation both within and among montane populations, with five to seven primary genetic lineages recovered. Multispecies coalescent validation analyses [guide tree and unguided Bayesian Phylogenetics and Phylogeography (BPP), Bayes factor delimitation (BFD)] using nuclear-only data congruently recover six or seven distinct lineages; BFD analyses using combined nuclear plus mitochondrial data favour seven or eight lineages. In stark contrast to this clear genetic fragmentation, a survey of secondary sexual features for available males indicates morphological conservatism across montane populations. While it is certainly possible that morphologically cryptic speciation has occurred in this taxon, this system may alternatively represent a case where extreme population genetic structuring (but not speciation) leads to an oversplitting of lineage diversity by multispecies coalescent methods. Our results have clear conservation implications for this federally endangered taxon and illustrate a methodological issue expected to become more common as genomic-scale data sets are gathered for taxa found in naturally fragmented habitats.