Intraspecific Molecular Variation Research Articles

Using intraspecific molecular and phenotypic variation to promote multi-functionality of reforestation during climate change – A review of tropical forest case studies in South-east Asia

Abstract The study of intraspecific genetic variation in plant traits for use in tropical forest restoration has broad potential for increasing our ability to achieve multi-functional objectives during this era of climate change. Developing seed-sourcing guidelines that optimize phenotypic characteristics best suited to a particular planting site as well as to future conditions imposed by environmental change could be useful for effective reforestation. Because evolution operates differently across tree species, this is an especially cumbersome task in tropical forests that contain thousands of species. Partially due to this high plant diversity, research and application of intraspecific variation in genetics, plant traits, and plant function in tropical forests wane far behind less diverse forest biomes. To examine the potential for improving reforestation efforts in tropical forests by considering intraspecific variation in plant traits and functions, we review the state of knowledge on intraspecific variation in South-east Asia as a case study. We focus on the dipterocarp family ( Dipterocarpaceae ), a highly diverse family of 16 genera with approximately 695 known species that often dominate lowland tropical rainforests of South-east Asia with many of these forests in a degraded state and in need of restoration. We found that there is research accumulating to understand genetic variation in approximately 10% of these 695 species. Intraspecific molecular variation exists at different spatial scales among species with 74% of species having moderate to high population differentiation (Fst > 0.10) and 92% of species with evidence of fine-scale genetic structure. Although this suggests a high potential for trait variation, few studies associated molecular with phenotypic variation. Seventeen tree species across 11 studies revealed intraspecific variation in traits or functions. Research indicates that intraspecific variation in growth may vary two-fold and drought tolerance four-fold among genotypes highlighting the possibility to pre-adapt trees to climate change during reforestation and to use intraspecific variation to promote the use of native species in commercial forestry. Our review presents opportunities and ideas for developing seed-sourcing guidelines to take advantage of intraspecific variation in traits and function by identifying how to locate this variation, which species would benefit, and how to test for trait variation. We also highlight an emerging area of research on local adaptation, common garden studies, and adaptive drought conditioning to improve reforestation during climate change.

Revisiting the bipolarity of Roaldia revoluta (Mitt.) P.E.A.S. Câmara & Carv.-Silva (Bryophyta, Pylaisiaceae)

AbstractThe occurrence of species in both polar regions (bipolarity) is a common phenomenon in the Antarctic flora. Considering the high morphological variation in polar regions due to extreme conditions, the use of molecular tools is indispensable for testing whether Arctic and Antarctic populations indeed belong to the same species. However, few phylogeographic studies of bipolar bryophytes have been conducted so far, especially when comparing molecular and morphological variation. Here, we assess the bipolarity and intraspecific variation of Roaldia revoluta, a strictly bipolar species of pleurocarpous mosses. Phylogenetic analyses based on ITS sequences clearly resolve R. revoluta as monophyletic and confirm its bipolar distribution pattern. Low intraspecific molecular variation in the markers ITS/26S and rpl16 was observed, and most specimens from both polar regions belong to a single haplotype, making it difficult to infer the origin and dispersal routes between both polar regions of R. revoluta. Morphometric analysis furthermore suggests that there are no significant morphological differences among populations from both polar regions and that morphological variation is mainly influenced by local environmental conditions. Our data do not unequivocally support the recent separation of the former intraspecific taxon Hypnum revolutum var. dolomiticum at the species level as Roaldia dolomitica.

Diversity assessment of Gloriosa superba accessions from Western Ghats of India based on morphological traits, ISSR markers and metabolite content

Gloriosa superba L. (glory lily) is a medicinally and horticulturally important plant used as a commercial source of the alkaloid colchicine. The Western Ghats in India is mostly unexplored for the morphological, genetic, and metabolite diversity of the species. In the current study, three methodologies were employed to assess the diversity among 50 established accessions of G. superba collected from several locations in the Western Ghats. The morphological analysis revealed that, among the studied traits, seed yield per vine and dry seed weight per 100 seeds for the established accessions were the most diverse traits. The former exhibited a significantly positive correlation with four vegetative and five yield traits. The first three principal components showed nine traits contributing to the variation. Intra-specific molecular variation based on inter simple sequence repeat markers distributed the accessions in three clusters with high polymorphism and low genetic diversity indices. Liquid chromatography high-resolution mass spectrometry analysis revealed significant variation among the screened accessions for colchicine content in tuber and seed tissue. Interestingly, an unexplored tissue, pod shell, was found to contain significantly high colchicine, which could be explored for commercial colchicine extraction. The morphogenetic and metabolite variation revealed by the study would be useful to develop elite varieties to expand the cultivation of glory lily across the country, fulfilling the increasing demand for colchicine and providing remunerative income to the farmers.

Intraspecific molecular variation among Androctonus crassicauda (Olivier, 1807) populations collected from different regions in saudi arabia

Androctonus crassicauda is one of the medically important scorpion species occurring in the Middle East. In this study, molecular variation in mtDNA cytochrome oxidase subunit I (COI) gene within seven populations of A. crassicauda from four main ecological regions of Saudi Arabia was investigated. Scorpion specimens were collected from different eco-geographical regions. DNA was extracted, subsequently 700 bp fragments of COI gene were amplified using specific scorpion primers and sequenced. The obtained sequences were analyzed, and phylogenetic trees were constructed. The phylogenetic analysis showed high levels of genetic variation among A. crassicauda populations with the existence of three distinct lineages. Also, it supports the existence of two distinct populations of A. crassicauda in Saudi Arabia, which perhaps suggestive of a putative distinct species. Further morphological studies with additional specimens from the Arabian Peninsula may reveal possible undiscovered and cryptic species in the region.

A New Relict Species of Slender Salamander (Plethodontidae: Batrachoseps) with a Tiny Range from Point Arguello, California

Among western North American amphibian lineages, the plethodontid salamander genus Batrachoseps has undergone the most extensive radiation. Here we describe a new species in the genus from the vicinity of Point Arguello, central California. This lineage falls within the B. pacificus group, but it is differentiated from other species in the group by both molecular sequence data and morphology. It is geographically disjunct from its close relatives, with a tiny range in unlikely habitat along a narrow strip of the Pacific Coast, where it is entirely surrounded by B. nigriventris, a distant relative. Although intraspecific molecular variation is almost entirely absent, some population structure was detected across the 4 km extent of its range. Because of its tiny range and limited genetic variation, the impacts of any potential modifications to its known habitat should be evaluated to ensure the species' continued conservation.

<strong><em>Cyathea</em> Sm. (Cyatheaceae) on Trindade Island (Brazil): An integrative approach</strong>

Trindade is a Brazilian oceanic island approximately 1,100 km from the American mainland. The Cyathea Sm. plants from Trindade were originally considered an endemic species, Cyathea copelandii Kuhn & Luerssen, but later included in Cyathea delgadii Sternb. Based on molecular data (trnL-trnF, trnG-trnR and rbcL-accD), morphometric analysis of leaf characters, scanning electron microscopy of indusia and spores, and habitat suitability modeling, the present study confirms the status of C. copelandii as a synonym of C. delgadii. Intraspecific molecular variation suggests phylogeographic structuring in C. delgadii and differentiation between the coastal Atlantic Forest domain and the Cerrado domain (savanna) in the interior of Brazil. The Trindade populations are more closely related to the Atlantic Forest populations, indicating a colonization of the island from the latter area.

Molecular phenotyping of maternally mediated parallel adaptive divergence within Rana arvalis and Rana temporaria.

When similar selection acts on the same traits in multiple species or populations, parallel evolution can result in similar phenotypic changes, yet the underlying molecular architecture of parallel phenotypic divergence can be variable. Maternal effects can influence evolution at ecological timescales and facilitate local adaptation, but their contribution to parallel adaptive divergence is unclear. In this study, we (i) tested for variation in embryonic acid tolerance in a common garden experiment and (ii) used molecular phenotyping of egg coats to investigate the molecular basis of maternally mediated parallel adaptive divergence in two amphibian species (Rana arvalis and Rana temporaria). Our results on three R.arvalis and two R.temporaria populations show that adaptive divergence in embryonic acid tolerance is mediated via maternally derived egg coats in both species. We find extensive polymorphism in egg jelly coat glycoproteins within both species and that acid-tolerant clutches have more negatively charged egg jelly - indicating that the glycosylation status of the jelly coat proteins is under divergent selection in acidified environments, likely due to its impact on jelly water balance. Overall, these data provide evidence for parallel mechanisms of adaptive divergence in two species. Our study highlights the importance of studying intraspecific molecular variation in egg coats and, specifically, their glycoproteins, to increase understanding of underlying forces maintaining variation in jelly coats.

Shared phylogeographic patterns between the ectocommensal flatworm Temnosewellia albata and its host, the endangered freshwater crayfish Euastacus robertsi.

Comparative phylogeography of commensal species may show congruent patterns where the species involved share a common history. Temnosewellia is a genus of flatworms, members of which live in commensal relationships with host freshwater crustaceans. By constructing phylogenetic trees based on mitochondrial COI and 28S nuclear ribosomal gene sequences, this study investigated how evolutionary history has shaped patterns of intraspecific molecular variation in two such freshwater commensals. This study concentrates on the flatworm Temnosewellia albata and its critically endangered crayfish host Euastacus robertsi, which have a narrow climatically-restricted distribution on three mountaintops. The genetic data expands upon previous studies of Euastacus that suggested several vicariance events have led to the population subdivision of Euastacus robertsi. Further, our study compared historical phylogeographic patterning of these species. Our results showed that phylogeographic patterns shared among these commensals were largely congruent, featuring a shared history of limited dispersal between the mountaintops. Several hypotheses were proposed to explain the phylogeographic points of differences between the species. This study contributes significantly to understanding evolutionary relationships of commensal freshwater taxa.

Phylogenetic relationships of Trachylepis skink species from Madagascar and the Seychelles (Squamata: Scincidae)

Lizards of the genus Trachylepis are a species-rich group of skinks mainly inhabiting Africa, Madagascar, and several other islands in the western Indian Ocean. All except one probably introduced species of Madagascan Trachylepis are endemic. Two species groups have been distinguished on the basis of subocular scale shape but their phylogenetic relationships remained unclear. We inferred a multilocus phylogeny of the Madagascan Trachylepis species, based on a concatenated dataset of 3261bp from 3 mitochondrial and 4 nuclear genes with a dense Madagascan taxon sampling and find high support for the monophyly of the endemic Madagascan Trachylepis. The two species groups in Madagascar are highly supported as clades. The highland species T. boettgeri is nested in the T. aureopunctata species group of mainly arid-adapted species, suggesting a colonization of highland swamps by ancestors inhabiting dry western Madagascar. The Seychellois species were sister to the T. maculilabris/T. comorensis clade, suggesting their origin directly out of Africa as with Seychellois chameleons. In Madagascar, a high intraspecific molecular variation was confirmed for T. gravenhorstii, T. elegans, and T. vato, indicating a need for taxonomic revision.

Population study of Atractolytocestus huronensis (Cestoda: Caryophyllidea), an invasive parasite of common carp introduced to Europe: mitochondrial cox1 haplotypes and intragenomic ribosomal ITS2 variants

The invasive monozoic tapeworm Atractolytocestus huronensis, a specific parasite of common carp, was originally found and described in the North American continent. It has been introduced to Europe and reported in several countries in the last 15years, as well. In the current study, tapeworms from one North American (USA) and five European localities (United Kingdom/UK, Slovakia, Hungary, Croatia, and Romania) were subjected to molecular analyses in order to determine the level of intrapopulation and intraspecific molecular variation and to assess interrelationships among American and European populations of the parasite. Partial sequences (672bp) of the mitochondrial cytochrome c oxidase subunit I (cox1) revealed the presence of only two cox1 haplotypes, in accordance with the nonnative character of the populations. The first haplotype was common for all tapeworms from the Continental Europe (Slovakia, Hungary, Croatia, and Romania); no differences were determined either within or among respective A. huronensis populations. The second cox1 haplotype was characterized in all individuals from the USA and UK, indicating their close genetic relationship. Both haplotypes differed in three nucleotide positions (99.6% identity) which did not change the amino acid sequence. The cox1 data imply that introduction of the parasite to Europe was probably the result of two independent events directed to the UK and Continental Europe. The very close genetic relationship between British and American A. huronensis was reflected also by similar ribosomal internal transcribed spacer 2 (ITS2) sequence structure; considerable intragenomic ITS2 variability was detected in all individuals of both geographic populations. Divergent ITS2 copies were mostly induced by different numbers of short repetitive motifs within the sequences, allowing their assortment into two ITS2 variants.

CULTIVATION-INDEPENDENT SPECIES IDENTIFICATION OF DINOBRYON SPECIES (CHRYSOPHYCEAE) BY MEANS OF MULTIPLEX SINGLE-CELL PCR1

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single-cell PCR (SC-PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.

Pseudo-cryptic species Arenicola defodiens and Arenicola marina (Polychaeta: Arenicolidae) in Wadden Sea, North Sea and Skagerrak: Morphological and molecular variation

The polychaete Arenicola defodiens, or black lug, was recently described as a morphologically highly similar species alongside the blow lug Arenicola marina. So far it was only known from the British Isles. A double spawning peak was observed earlier in lugworms of the western Wadden Sea. Here, we test the hypothesis that the two spawning peaks represent the two species in sympatry. This hypothesis is refuted on the basis of both morphological and mitochondrial DNA data; both spawning peaks are attributed to A. marina. In spite of this, we confirm, on the basis of new collections as well as re-examination of museum collections, the presence of A. defodiens in the western Wadden Sea, North Sea and also the Skagerrak; its distribution is restricted to habitats which are either submerged or have short emersion times, have relatively coarse sediments and high and stable salinities. Sympatry is common. The species differ strongly in the mitochondrial DNA fragment examined; the observed 14% uncorrected minimum difference amounts to an estimated 33–63 million years since sequence divergence. The amount of intraspecific molecular variation is larger for A. defodiens than for A. marina. This is evidence to suggest that A. marina may have undergone more recent and/or more severe population size bottlenecks.

Phylogeographic evidence for the existence of an ancient biogeographic barrier: the Isthmus of Kra Seaway

Biogeographic boundaries are characterised by distinct faunal and floral assemblages restricted on either side, but patterns among groups of taxa often vary and may not be discrete. Historical biogeography as a consequence, while providing crucial insights into the relationship between biological diversity and earth history, has some limitations. Patterns of intraspecific molecular variation, however, may show unambiguous evidence for such historical divides, and can be used to test competing biogeographic hypotheses (often based on the dispersal-vicariance debate). Here, we utilise this method to test the hypothesis that a major biogeographic transition zone between the Sundaic and Indochinese biotas, located just north of the Isthmus of Kra in SE Asia, is the result of Neogene marine transgressions that breached the Isthmus in two locations for prolonged periods of time (>1 million year duration). Phylogeographic analyses of a freshwater decapod crustacean, the giant freshwater prawn Macrobrachium rosenbergii, strongly supports the historical existence of the more northerly postulated seaway. Results presented here highlight the power of utilising intraspecific molecular variation in testing biogeographical hypotheses.

Evolution of developmental genes: molecular microevolution of enhancer sequences at the Ubx locus in Drosophila and its impact on developmental phenotypes.

Homeotic genes, which function to specify segment identity along the anterior-posterior axis of embryos, are controlled by extensive batteries of enhancer sequences. We have investigated patterns of interspecific and intraspecific molecular variation in three enhancers of the Ultrabithorax (Ubx) locus, which are bx-32.8, pbx32.7, and bxd4.1, from the Drosophila melanogaster species group. These enhancer sequences control Ubx expression by binding to multiple transcription factors encoded by gap, pair-rule, and dorsoventrally expressed genes. Sequence comparisons reveal purifying selection acting on all three enhancers, both in bases binding transcription factors and in bases whose functions are as yet unknown. Neutrality tests largely fail to reject a neutral evolution model. However, using a matrix similarity value to reflect the binding affinity of the protein-binding sites, interspecific and intraspecific variation that may have potential to affect the binding affinity of the sequences homologous to those binding transcription factors in D. melanogaster are discovered, suggesting evolutionary flexibility in the way in which these sequences function in the control of development. As a means of measuring the impact of intraspecific variation on observable phenotypes, we have induced Ubx mutant phenocopies with embryonic ether treatment, and find strong and highly significant variation between D. melanogaster strains in their phenocopy frequencies. This variation shows no significant correlation with the strengths of the mutant phenotypes when the strains are heterozygous with a Ubx null mutation. Estimated phylogenetic trees have been constructed for the three enhancer regions investigated. Neither of the two phenotypic traits investigated shows any significant associations with the phylogeny of any of the three enhancers.

Intraspecific molecular variation in the seaweed fly Coelopa frigida consistent with behavioural distinctness of British and Swedish populations.

The major aim of this study was to compare the intraspecific variation and genetic structure of the behaviourally distinct British and Swedish populations of the seaweed fly Coelopa frigida. C. frigida has been the subject of intense study into the basis of female choice. The behaviour of British females is consistent with a 'good genes' model, whereas that of the Swedish flies suggests a Fisher process, in which the difference between the former and the latter is defined by female choice increasing offspring viability in 'good genes' models. Through a study of variability in the mitochondrial cytochrome oxidase II gene from more than 600 flies, we show that there is clear differentiation at the molecular level between the two countries' populations, with an FST of > 75% and no shared haplotypes. Tajima's test reveals an excess of rare variants relative to expectation, which, if not the result of selective sweep, indicates either a population expansion or purifying selection against weakly deleterious variants. Within the two populations, substantial subpopulation differentiation is observed in the UK, where there is also evidence of isolation by distance. Swedish populations exhibit lower variability, and no evidence of isolation by distance, with the latter result possibly being related to the continuous distribution of suitable habitat. The pattern of intraspecific variation is explainable by a combination of contemporary and also historical factors. British and Swedish populations may have been descended from at least two separate founding populations during the recolonization of these areas following Pleistocene glaciations.

Source regions for recruitment of Calanus finmarchicus to Georges Bank: evidence from molecular population genetic analysis of mtDNA

Molecular population genetic analysis has provided evidence that the copepod, Calanus finmarchicus, of the Labrador Current, Gulf of St Lawrence, Scotian Shelf, Gulf of Maine, and Georges Bank constitute a single, interbreeding population. The DNA sequence of a 350 base pair portion of the mitochondrial large subunit (16S) ribosomal RNA (rRNA) gene was determined for a total of 72 individuals collected in 1992, and 110 individuals collected in 1993 from these regions. There was significant heterogeneity in haplotype frequencies among the samples collected in 1992, but this heterogeneity did not resolve into regional patterns. The only regional differences seen were between pooled samples of the western N. Atlantic and those of the Norwegian Sea. There were no significant differences in haplotype frequencies among the samples collected in 1993, and fewer haplotypes were observed in these samples. Intraspecific molecular variation was typical of other marine species: there were 29 haplotypes among the 182 individuals sequenced. The frequency distribution of the haplotypes was highly skewed: 128 individuals shared one haplotype and 19 individuals were unique. There were 24 variable sites among the 350 bases sequenced; estimated nucleotide diversity was 0.0042. The genetic character of C. finmarchicus populations in the western N. Atlantic was stable over time in that three of the haplotypes (including the most abundant) occurred in both 1992 and 1993. However, haplotype frequencies differed significantly between the two years. The lack of regional structure in the 1992 samples and the genetic homogeneity of samples collected in 1993 across the domain from the Labrador Current to the Gulf of St Lawrence to Georges Bank and the Gulf of Maine indicated that there is significant gene flow across this region. The persistent genetic pattern suggests that the Gulf of St Lawrence may be an important source region for recruitment of C. finmarchicus to Georges Bank. Determination of zooplankton dispersal patterns within high gene flow species will provide information that may not be determined by conventional oceanographic analyses.