Evolutionary distinctiveness and conservation priority of the endangered Najas ancistrocarpa (Hydrocharitaceae)

Phylogeny is vitally important to evaluate evolutionary distinctiveness and conservation priorities of species. Najas ancistrocarpa is one of the rarest and least known East Asian species of the c. 30–40 species of the aquatic plant genus Najas (Hydrocharitaceae). We used a recently discovered collection of this nationally and regionally endangered species in Japan and East Asia to assess whether N. ancistrocarpa is a distinct species or an untypical form of other Najas species and to evaluate its conservation priority in a phylogenetic context. The sample set of our previous study, exploring worldwide phylogeny of the genus was used with N. ancistrocarpa to delimit species boundaries in Najas and obtain conservation priority scores, calculated by multiplying evolutionary distinctiveness and global extinction risks converted from Red List categories. We performed molecular phylogenetic analyses of plastid (ptDNA) and nuclear (ITS) DNA datasets, STACEY species delimitation analysis using the multilocus dataset and a measurement of conservation priority with Tuatara. Najas ancistrocarpa was consistently placed in ptDNA and ITS trees. Species delimitation analysis objectively assessed that N. ancistrocarpa is phylogenetically distinct. Phylogenetic conservation prioritization ranked N. ancistrocarpa the second highest priority species of the genus Najas. Overall, in terms of biodiversity conservation, N. ancistrocarpa represents an important Najas species in Japan and probably the world.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

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.     

Population and phylogenomic decomposition via genotyping‐by‐sequencing in Australian Pelargonium

Species delimitation has seen a paradigm shift as increasing accessibility of genomic‐scale data enables separation of lineages with convergent morphological traits and the merging of recently diverged ecotypes that have distinguishing characteristics. We inferred the process of lineage formation among Australian species in the widespread and highly variable genus Pelargonium by combining phylogenomic and population genomic analyses along with breeding system studies and character analysis. Phylogenomic analysis and population genetic clustering supported seven of the eight currently described species but provided little evidence for differences in genetic structure within the most widely distributed group that containing P. australe. In contrast, morphometric analysis detected three deep lineages within Australian Pelargonium; with P. australe consisting of five previously unrecognized entities occupying separate geographic ranges. The genomic approach enabled elucidation of parallel evolution in some traits formerly used to delineate species, as well as identification of ecotypic morphological differentiation within recognized species. Highly variable morphology and trait convergence each contribute to the discordance between phylogenomic relationships and morphological taxonomy. Data suggest that genetic divergence among species within the Australian Pelargonium may result from allopatric speciation while morphological differentiation within and among species may be more strongly driven by environmental differences.     

Evolutionary history and systematics of Acer section Acer – a case study of low-level phylogenetics

The phylogenetic and systematic position of all species of Acer section Acer from North America, East Asia, and western Eurasia are evaluated using various splits-based networks (distance networks, bipartition networks), ITS motif analysis, and morphology. Molecular analyses are based on 276 ITS clones obtained from 101 specimens collected mainly from natural stands. The large sample size ensures to cover sufficiently inter- and intraspecific ITS variability of this group. Formerly recognised species are generally supported by ITS data and morphology; the combination of molecular (ITS) and morphological criteria allows defining seven (supraspecific) taxonomic groups prior to a phylogenetic reconstruction. Phylogenetic signals captured in modern ITS sequences are partly incompatible but clearly suggest that Acer section Acer underwent three major radiations. Horizontal gene flow is indicated between ancestors of extant taxa that are isolated at present times. The level of ITS derivation can be estimated and corresponds to levels of morphological differentiation and (palaeo-) biogeographical patterns. Based on our results we question the potential of cladistic approaches to infer low-level evolution in an adequate manner and demonstrate that speciation in members of Acer section Acer is not generally linked to cladogenesis. The data and methodologies provided here allow to trace pathways of low-level evolution and to analyse such data sets with a less restricted (non-dichotomous) dynamic concept. Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Species delimitation and phylogeography of the Abies chensiensis complex inferred from morphological and molecular data

Species delimitation in the Abies chensiensis complex, consisting of A. chensiensis, A. recurvata, A. ernestii and A. ernestii var. salouenensis, has been the subject of a long‐term dispute, as various taxonomic combinations have been proposed. We combined different lines of evidence, including morphological characters and mitochondrial and plastid DNA sequence data, to assess species delimitation. Plastid DNA sequence variation was highly consistent with the morphological result and separated A. recurvata from the other three taxa, but the mitochondrial DNA genealogy was more strongly related to geographical distribution rather than species delimitation, as proposed in previous studies. On the basis of morphological characters and plastid DNA sequence variation, the current species status of A. recurvata was well supported, and we also accepted A. ernestii and A. ernestii var. salouenensis as varieties of A. chensiensis. Furthermore, the phylogeographical history of A. chensiensis was surveyed by molecular data and ecological niche modelling. On the basis of molecular data, the current phylogeographical pattern of the A. chensiensis complex was probably shaped by recent rapid expansions from multiple refugia in the Hengduan Mountains and habitat contraction after the last glaciation. This hypothesis was supported by the results of ecological niche modelling and previous fossil records. Our findings suggest that these fir taxa might have continued to expand their range after the largest glaciation until the end of the last glaciation, but have contracted since then with the increasing temperature and habitat changes. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 175–188.     

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.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.     

Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms

A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed intrageneric classifications were evaluated. The phylogenetic tree showed that (1) sectionsArguta, Cissifolia, Lithocarpa, Macrantha, Palmata, Spicata, Tataricum, Trifoliata sensu Ogata (1967: Bull. Tokyo Univ. Forests 63: 89–206) were monophyletic groups respectively, (2) sectionsCampestria, Goniocarpa, Platanoidea sensu Ogata (1967) were polyphyletic respectively, and (3) sectionsDistyla andParviflora formed a sister group. An average of estimated nucleotide substitution rates of Acer chloroplast DNA was calculated as 7.9×10⁻¹¹±1.4×10⁻¹¹ nucleotide substitutions par site par year, which coincides well with previously reported rates of perennial plants. Divergence eras of eastern Asia and North American species in both sectionsSpicata andRubra were estimated to be late Miocene. In consideration with previous data, multiple migrations and disjunctions are likely to have formed the eastern Asian and North American disjunct distribution.     

Spatio-ecological niche segregation of two sympatric species of<Emphasis Type="Italic">Clidemia (Melastomataceae)</Emphasis> in Western Amazonian non-flooded rainforests

The frequent occurrence of sympatric series of closely related plant species in tropical rainforests has evoked claims for and against the application of the competitive exclusion principle in these ecosystems. Narrow niche limits defined by biotic as well as abiotic specialization have been reported for sympatric species of the same genus or family. In Amazonian lowland rainforests this question deserves renewed attention because: (1) the existence of edaphically defined community types has recently been well established, and (2) spatio-ecological niche segregation of congeneric species may help explain not only the maintenance of the high Amazonian alpha-diversity, but also its origin through sympatric ecological speciation. In this study, the morphology, ecology, and distribution patterns of two species,Clidemia epiphytica andC. longifolia (Melastomataceae), from western Amazonia, were analyzed. The aims were to find out whether they really are two distinct taxonomic species and if so, whether they also can be considered biological species; if the species are sympatric; and if they are ecologically specialized. The results showed that the morphological variation of the species seems continuous, but that they exhibit opposite morphological responses to variation in soil cation concentration, which suggests that they also are separate biological species. Furthermore, the species occur sympatrically but in different habitats. It is suggested that a part of the enigma of sympatric congeners in rainforests may be explainable by spatial segregation stemming from ecological specialization in relation to subtle environmental variation. It is hypothesized that the studied species are a good candidate case of sympatric speciation driven by ecological specialization.     

Species delimitation in annual killifishes from the Brazilian Caatinga,the Hypsolebias flavicaudatus complex (Cyprinodontiformes: Rivulidae): implications for taxonomy and conservation

The organisms investigated are members of the Hypsolebias flavicaudatus species complex; a clade of morphologically similar annual killifishes endemic to the semi-arid savannah of north-eastern Brazil in the São Francisco River basin, which is a tropical area under intensive process of habitat loss. A taxonomic revision was conducted combining two different approaches for species delimitation – a tree-based approach using mt-DNA (cytochrome b) and a character-based approach using morphological variation (colour patterns, fin morphology, meristic and morphometric data). Nine species are recognised, of which five are new species: H. flagellatus (Costa), H. flavicaudatus (Costa & Brasil), H. gilbertobrasili sp. nov., H. guanambi (Costa & Amorim), H. janaubensis (Costa), H. nitens sp. nov., H. pterophyllus sp. nov., H. radiseriatus sp. nov. and H. sertanejo sp. nov. On the basis of 17 years of field studies, preliminary conservation status assessments indicate that two species are highly threatened with extinction, at least one is endangered and another is vulnerable, whereas the others are not endangered. This result deeply contrasts with a previous assessment 10 years ago, when the whole H. flavicaudatus complex was considered as a single species of no conservation concern. Discrepancies in the assessment of conservation among closely related species living in similar habitats are a consequence of heterogeneous human occupation and disproportionate recent development throughout the São Francisco River basin.     

Ecology and Hybridization Potential of Two Sympatric Metallophytes,the Narrow Endemic Crepidorhopalon perennis (Linderniaceae) and its More Widespread Congener C. tenuis

Crepidorhopalon perennis is a metallophyte critically endangered by mining activities and currently known from only one site on copper‐rich soils in Katanga (Dem. Rep. Congo). It is closely related to the annual C. tenuis, also a rare metallophyte, but with a broader geographical range. We investigated the variation in morphometric traits and ecological niches (based on edaphic conditions and pollinator assemblages) of C. perennis and C. tenuis, to evaluate the risk of inter‐specific competition, and their potential for hybridization to ascertain if C. perennis might be at risk of genetic swamping by its more widespread congener. We examined whether species were found under sympatric or parapatric settings with opportunity for hybridization (or gene exchange). Such knowledge is essential for implementing restoration management protocols, including the introduction of C. perennis into substitution sites where C. tenuis might be already present. Fourteen morphological characters and 11 soil variables were measured and visiting pollinator species were identified at the site where the two species co‐occur. Our results show that the two species can be distinguished based on their morphological traits, show niche overlap based on edaphic properties, and share the same pollinator assemblage. In addition, no morphologically intermediate individuals could be detected, suggesting no hybridization, and that the two species may be reproductively isolated. We conclude that C. perennis conservation and restoration operations can be realized in substitution sites where C. tenuis may be present, with the need, however, to evaluate the potential effect of sharing a pollinator assemblage on reproductive success of both species.     

Reproductive ecology of three endangered African violet (Saintpaulia H. Wendl.) species in the East Usambara Mountains, Tanzania

Knowledge of the reproductive biology of endangered plants is essential for their effective conservation. It also provides important information for understanding the evolutionary processes that affect speciation, thus helping the definition of proper units for conservation in endangered plants with problematic taxonomy. We studied the reproductive potential and possibility for hybridization in the endangered genus Saintpaulia (Gesneriaceae) by examining flowering phenology, flower and seed production and pollination of three sympatric cross‐compatible Saintpaulia species in the East Usambara Mts., Tanzania. The synchrony observed in flowering in S. confusa and S. difficilis may enable hybridization between these two species, whereas partial phenological separation may contribute to the integrity of S. grotei. Although the level of flower abortion is high in S. confusa, each pollinated flower yields about 1000 seeds. Saintpaulia confusa produces fruits following both self‐ and cross‐pollination but spontaneous self‐pollination seems not to occur. Thus, seed production depends on sufficient pollinator service. Floral heteromorphy (i.e. enantiostyly) and bee pollination are likely to further enhance cross‐pollination, suggesting that the genus predominantly outcrosses. Thus, Saintpaulia populations are likely to suffer from negative effects of inbreeding if they become small and isolated.     

Highlighting convergent evolution in morphological traits in response to climatic gradient in African tropical tree species: The case of genus Guibourtia Benn.

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Delimiting cryptic pathogen species causing apple Valsa canker with multilocus data

Fungal diseases are posing tremendous threats to global economy and food safety. Among them, Valsa canker, caused by fungi of Valsa and their Cytospora anamorphs, has been a serious threat to fruit and forest trees and is one of the most destructive diseases of apple in East Asia, particularly. Accurate and robust delimitation of pathogen species is not only essential for the development of effective disease control programs, but also will advance our understanding of the emergence of plant diseases. However, species delimitation is especially difficult in Valsa because of the high variability of morphological traits and in many cases the lack of the teleomorph. In this study, we delimitated species boundary for pathogens causing apple Valsa canker with a multifaceted approach. Based on three independent loci, the internal transcribed spacer (ITS), β‐tubulin (Btu), and translation elongation factor‐1 alpha (EF1α), we inferred gene trees with both maximum likelihood and Bayesian methods, estimated species tree with Bayesian multispecies coalescent approaches, and validated species tree with Bayesian species delimitation. Through divergence time estimation and ancestral host reconstruction, we tested the possible underlying mechanisms for fungal speciation and host‐range change. Our results proved that two varieties of the former morphological species V. mali represented two distinct species, V. mali and V. pyri, which diverged about 5 million years ago, much later than the divergence of their preferred hosts, excluding a scenario of fungi–host co‐speciation. The marked different thermal preferences and contrasting pathogenicity in cross‐inoculation suggest ecological divergences between the two species. Apple was the most likely ancestral host for both V. mali and V. pyri. Host‐range expansion led to the occurrence of V. pyri on both pear and apple. Our results also represent an example in which ITS data might underestimate species diversity.     

Trophic niche similarities of sympatric Turdus thrushes determined by fecal contents,stable isotopes,and bipartite network approaches

An ecological niche has been defined as an n‐dimensional hypervolume formed by conditions and resources that species need to survive, grow, and reproduce. In practice, such niche dimensions are measurable and describe how species share resources, which has been thought to be a crucial mechanism for coexistence and a major driver of broad biodiversity patterns. Here, we investigate resource partitioning and trophic interactions of three sympatric, phylogenetically related and morphologically similar species of thrushes (Turdus spp.). Based on one year of data collected in southern Brazil, we investigated niche partitioning using three approaches: diet and trophic niche assessed by fecal analysis, diet and niche estimated by stable isotopes in blood and mixing models, and bipartite network analysis derived from direct diet and mixing model outputs. Approaches revealed that the three sympatric thrushes are generalists that feed on similar diets, demonstrating high niche overlap. Fruits from C3 plants were one of the most important food items in their networks, with wide links connecting the three thrush species. Turdus amaurochalinus and T. albicollis had the greatest trophic and isotopic niche overlap, with 90% and 20% overlap, respectively. There was partitioning of key resources between these two species, with a shared preference for fig tree fruits—Ficus cestrifolia (T. amaurochalinus PSIRI% = 11.3 and T. albicollis = 11.5), which was not present in the diet of T. rufiventris. Results added a new approach to the network analysis based on values from the stable isotope mixing models, allowing comparisons between traditional dietary analysis and diet inferred by isotopic mixing models, which reflects food items effectively assimilated in consumer tissues. Both are visualized in bipartite networks and show food‐consumers link strengths. This approach could be useful to other studies using stable isotopes coupled to network analysis, particularly useful in sympatric species with similar niches.     

An integrative approach to delimiting species in a rare but widespread mycoheterotrophic orchid

In the spirit of recent calls for species delimitation studies to become more pluralistic, incorporating multiple sources of evidence, we adopted an integrative, phylogeographic approach to delimiting species and evolutionarily significant units (ESUs) in the Corallorhiza striata species complex. This rare, North American, mycoheterotrophic orchid has been a taxonomic challenge regarding species boundaries, displaying complex patterns of variation and reduced vegetative morphology. We employed plastid DNA, nuclear DNA and morphometrics, treating the C. striata complex as a case study for integrative species delimitation. We found evidence for the differentiation of the endangered C. bentleyi (eastern USA) + C. striata var. involuta (Mexico) from the remaining C. striata (= C. striata s.s.; USA, Canada, Mexico). Corallorhiza striata involuta and C. bentleyi, disjunct by thousands of kilometres (Mexico‐Appalachia), were genetically identical but morphologically distinct. Evidence suggests the C. striata complex represents three species: C. bentleyi, C. involuta and a widespread C. striata s.s under operational criteria of diagnosability and common allele pools. In contrast, Bayesian coalescent estimation delimited four species, but more informative loci and a resultant species tree will be needed to place higher confidence in future analyses. Three distinct groupings were identified within C. striata s.s., corresponding to C. striata striata, C. striata vreelandii, and Californian accessions, but these were not delimited as species because of occupying a common allele pool. Each comprises an ESU, warranting conservation considerations. This study represents perhaps the most geographically comprehensive example of integrative species delimitation for any orchid and any mycoheterotroph.     

When morphological identification meets genetic data: the case of Lucanus cervus and L. tetraodon (Coleoptera,Lucanidae)

The European stag beetle Lucanus cervus, widely distributed across Europe and in some Near East countries, is one of the best known coleopteran species listed in the European Habitats Directive 92/43/EEC and it is considered a flagship species for conservation of saproxylic fauna. Lucanus tetraodon is a closely related species whose geographical distribution is still poorly known and debated. The two species have a sympatric occurrence in central Italy, and in some localities of these areas, many individuals show a mosaic of morphological traits that makes species assignment nearly impossible. We used both mitochondrial and nuclear markers to analyse these specimens. The mitochondrial results evidenced that the two species represent well‐defined genetic entities with mitochondrial DNA introgression. This pattern could be the result of either hybridization or of a convergence of morphological characters under local selective pressures in areas of sympatric occurrence. The nuclear marker was polymorphic across the two species and therefore did not reveal hybridization, even if many are the supports to this phenomenon. The most plausible explanation for this genetic pattern is a very recent divergence of the two species which share a common origin and thus a common wg genotype.