Assessing hidden species diversity in the coral Pocillopora damicornis from Eastern Australia

The incredible range of morphological plasticity present in scleractinian corals has confused the taxonomy of the group, prompting the introduction of “ecomorphs” to explain the observed correlation between local environmental conditions and phenotypic variation. Pocillopora damicornis (Linnaeus, 1758) represents one of the best known examples of eco-phenotypic variation in scleractinian corals with a variety of forms and reproductive strategies reported across its global distribution range. Here, we reconstruct genealogical relationships of P. damicornis colonies collected from thirteen locations along the East Australian coast to examine the relationship between genetic and phenotypic diversity in this species. Haplotype networks computed from two mitochondrial DNA regions (CR, ORF) indicate that the range of morphotypes observed within this taxon fall into at least five genetically distinct mitochondrial lineages. Nuclear (HSP70, ITS2) haplowebs on the other hand recover sharp genetic discontinuities among three of the morphological groups. We conclude that P. damicornis from Eastern Australia constitutes a cryptic species complex. The misinterpretation of taxonomical units within P. damicornis may well explain its perceived variation in the ecology, biology and life history across its range.     

Molecular identification of symbiotic dinoflagellates in Pacific corals in the genus <Emphasis Type="Italic">Pocillopora</Emphasis>

This study focused on the association between corals of the genus Pocillopora, a major constituent of Pacific reefs, and their zooxanthellae. Samples of P. meandrina, P. verrucosa, P. damicornis, P. eydouxi, P. ligulata and P. molokensis were collected from French Polynesia, Tonga, Okinawa and Hawaii. Symbiodinium diversity was explored by looking at the 28S and ITS1 regions of the ribosomal DNA. Most zooxanthellae were found to belong to clade C, sub-clade C1, with little differentiation between populations. Interestingly, individuals of P. damicornis harbored sub-clade C1, clade D and clade A, depending on location. The symbiotic association of P. damicornis with its zooxanthellae may be somewhat more flexible than those of other Pocillopora species.     

A fluorescence-based quantitative real-time PCR assay for accurate <Emphasis Type="Italic">Pocillopora damicornis</Emphasis> species identification

Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.     

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.     

Long forsaken species diversity in the Middle American lizard Holcosus undulatus (Teiidae)

Numerous reptile species have been divided into subspecies. Although this classification may capture the morphological variation within species, it often conceals significant species diversity because many subspecies actually represent species under lineage‐based species concepts. The lizard Holcosus undulatus is a common, widely distributed, monotypic species in Middle America. However, 12 subspecies of this taxon were recognized until the early 1970s. We used two lineage‐based methods for species delimitation to re‐evaluate species limits within H. undulatus with DNA sequence and morphological data. We included all the previously recognized subspecies of H. undulatus except H. u. miadis. Holcosus undulatus was exclusive. In addition, H. u. amphigrammus, H. u. gaigeae, H. u. hartwegi, H. u. parvus, H. u. pulcher, H. u. sinister, H. u. stuarti, H. u. thomasi and H. u. undulatus were supported as distinct evolutionary lineages based on the molecular and morphological evidence. We therefore elevate all of these subspecies to species rank. In addition, two separate mitochondrial lineages may represent cryptic, undescribed species within H. undulatus. The morphological distinctness and allopatry of H. u. miadis and H. u. pulcher, as well as the high genetic divergence of the latter species, suggest that they also represent distinct evolutionary species. Our results also suggest that additional species diversity may still be hidden within the H. u. amphigrammus, H. u. parvus, H. u. sinister and H. u. undulatus lineages. This work supports resurrection of overlooked diversity within Holcosus, which has important implications for the conservation of this genus in Middle America. © 2015 The Linnean Society of London     

Species and population genomic differentiation in Pocillopora corals (Cnidaria,Hexacorallia)

Correctly delimiting species and populations is a prerequisite for studies of connectivity, adaptation and conservation. Genomic data are particularly useful to test species differentiation for organisms with few informative morphological characters or low discrimination of cytoplasmic markers, as in Scleractinians. Here we applied Restriction site Associated DNA sequencing (RAD-sequencing) to the study of species differentiation and genetic structure in populations of Pocillopora spp. from Oman and French Polynesia, with the objectives to test species hypotheses, and to study the genetic structure among sampling sites within species. We focused here on coral colonies morphologically similar to P. acuta (damicornis type β). We tested the impact of different filtering strategies on the stability of the results. The main genetic differentiation was observed between samples from Oman and French Polynesia. These samples corresponded to different previously defined primary species hypotheses (PSH), i.e., PSHs 12 and 13 in Oman, and PSH 5 in French Polynesia. In Oman, we did not observe any clear differentiation between the two putative species PSH 12 and 13, nor between sampling sites. In French Polynesia, where a single species hypothesis was studied, there was no differentiation between sites. Our analyses allowed the identification of clonal lineages in Oman and French Polynesia. The impact of clonality on genetic diversity is discussed in light of individual-based simulations.

     

Broadcast Spawning by Pocillopora Species on the Great Barrier Reef

The coral genus Pocillopora is one of the few to include some species that broadcast spawn gametes and some species that brood larvae, although reports of reproductive mode and timing vary within and among species across their range. Notably, the ubiquitous Pocillopora damicornis has been described as both a brooder and spawner, although evidence of broadcast spawning is rare. Here, we report observations of broadcast-spawning in four species of Pocillopora on the Great Barrier Reef (GBR), including P. damicornis. All species spawned predictably during the early morning, two days following the full moon, and spawning was observed in multiple months over the summer period (November to February). Eggs and sperm were free-spawned concurrently. Eggs were negatively buoyant and contained Symbiodinium. This newfound knowledge on the mode, timing and regularity of broadcast spawning in Pocillopora spp. on the GBR brings us one step closer to elucidating the complex reproductive ecology of these species.     

DNA barcoding supports reclassification of Japanese Chironomus species (Diptera: Chironomidae)

Non‐biting midges (Diptera: Chironomidae) adapt to species‐specific environmental conditions and hence are promising bioindicators for aquatic and ecotoxicological monitoring. Although their utility for these purposes was historically limited by difficulties in their morphological identification, DNA barcoding offers a possible solution. Here, eight Japanese species of the genus Chironomus, which is characterized by its worldwide distribution and abundance among Chironomidae, were subjected to DNA barcoding using cytochromec oxidase subunit I (COI). To examine whether this DNA barcode is a useful indicator for Japanese species of Chironomus, we calculated genetic distances within and between the COI sequences of Chironomus species both from this study and worldwide and constructed phylogenetic trees. Based on 415 bp COI sequences and the Kimura two‐parameter model, the average genetic distances within 37 species and between 72 species were 2.6% and 17.2%, respectively. Although the ranges of genetic distances within and between species overlapped from 0.8% to 17.3%, 99.7% of average genetic distances between species were >3.0%. Some of this overlap is attributable to distances within species that were “too large” as well as those between species that were “too small”. Of eight Japanese species examined, two showed genetic distances between species that were below a 3.0% threshold, and four had distances within species that were greater than 3.0%. These results suggest a possible reclassification of these species and the need for further sampling to unveil biogeographic variations among different countries and regions.     

Morphological variation inLolium (Poaceae) as a measure of species relationships

Analysis of morphological and phenological data for determining the genetic variation within sevenLolium species led to the recognition of two groups within this genus. One group, containing the two inbreeding speciesL. temulentum andL. persicum, was clearly distinct from all other species. Strong morphological and phenological intergradation was found between both species. The cross-breeding species,L. perenne, L. rigidum, andL. multiflorum, formed another group. Little differentiation was found between these species, though they were distinct. Two inbreeding species,L. loliaceum andL. remotum, were clearly distinct from each other and the two groups.L. loliaceum had an isolated position and was most related toL. rigidum. L. remotum had an intermediate position between the cross-breeding and inbreeding species, and was almost equally distant from all three cross-breeding species.Genetic variation inLolium spp. I.     

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.     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.     

Isolation and characterization of microsatellite loci in Penstemon rostriflorus (Plantaginaceae) and cross‐species amplification

Eight novel microsatellite primer pairs are presented for Penstemon rostriflorus, representing the first microsatellite markers available for this genus. Loci were characterized for 20 individuals from two populations in the Great Basin, USA. All loci are polymorphic within P. rostriflorus (seven to 13 alleles per locus; observed heterozygosity between 0.40 and 0.95), and therefore useful for population genetic studies within the species. Cross‐species transferability was tested on 40 additional species of Penstemon, and results indicate that these primers pairs will likely be useful for population genetic studies on many Penstemon species.     

Pallenopsis patagonica (Hoek, 1881) – a species complex revealed by morphology and DNA barcoding,with description of a new species of Pallenopsis Wilson, 1881

Pallenopsis patagonica (Hoek, 1881) is one of the most taxonomically problematic and variable pycnogonid species, and is distributed around the southern South American coast, and the Subantarctic and Antarctic areas. We conducted a phylogenetic analysis of mitochondrial cytochrome c oxidase subunit I (COI) sequences of 47 Pallenopsis specimens, including 39 morphologically identified as P. patagonica, five Pallenopsis pilosa (Hoek, 1881), one Pallenopsis macneilli Clark, 1963, one Pallenopsis buphtalmus Pushkin, 1993, and one Pallenopsis latefrontalis Pushkin, 1993. Furthermore, we studied morphological differences between the different COI lineages using light and scanning electron microscopy, including also material from Loman's and Hedgpeth's classical collections, as well as Hoek's type material of P. patagonica from 1881. The molecular results unambiguously reveal that P. patagonica is a complex of several divergent clades, which also includes P. macneilli, P. buphtalmus, and P. latefrontalis. Based on the material available, two major clades could be identified, namely a ‘Falkland’ clade, to which we assign the nominal P. patagonica, and a ‘Chilean’ clade, which is distinct from the ‘Falkland’ clade. We describe the ‘Chilean’ clade as new species, P allenopsis yepayekae sp. nov. Weis, 2013. All molecular results are confirmed by specific morphological characteristics that are discussed in detail and compared with Pallenopsis species closely related to the P. patagonica complex. Our results reveal that P. patagonica is a species‐rich complex that is in need for a thorough taxonomic revision, using both morphological and genetic approaches. © 2014 The Linnean Society of London