Genetic identification and hybridization in the seagrass genus Halophila (Hydrocharitaceae) in Sri Lankan waters

Abstract

Seagrasses, as marine angiosperms, play important roles in coastal ecosystems. With increasing anthropogenic impacts, they are facing dramatic declines on a global scale. Halophila is well-known as a complex taxonomic challenge mainly due to high morphological plasticity. By using only a morphological approach, the genus could be over-split or similar species could be erroneously lumped, thus masking its true biodiversity. In the present study, we incorporated genetic identification with morphological examination to reveal the identity of Halophila plants in southern and northwestern Sri Lankan waters. The nuclear ribosomal internal transcribed spacer (ITS) region and chloroplast ribulose-bisphosphate carboxylase gene (rbcL) were used to identify plants collected from the Gulf of Mannar, Puttalam Lagoon, and Matara, Sri Lanka. Based on genetic identification, H. major (Zoll.) Miquel is reported for the first time from Sri Lanka, which might have been misidentified as H. ovalis in previous literature based on morphology alone. We also observed a first hybridization case of Halophila cross between H. ovalis and H. major. Two potential cryptic species were found, herein designated Halophila sp. 1 (allied to H. minor) and Halophila sp. 2 (closely related to H. decipiens). In order to clarify taxonomic ambiguity caused by morphological plasticity and the low resolution of genetic markers, further comparative phylogenomic approaches might be needed to solve species boundary issues in this genus.

Keywords: DNA barcoding; Hybridization; ITS; Morphological plasticity; Species boundary; rbcL.

Figure 1. Sample collection sites coded with different colors (blue: Mannar, green: Kalpitiya, and red: Matara) for Halophila surveys in Sri Lanka. Numbers in brackets indicate sample size.

Figure 2. Comparison of leaf morphology of H. major, H. ovalis, and H. stipulacea like specimens collected in Sri Lanka. Samples displayed in this figure were included and showed in ITS tree (Fig. 3).

Figure 3. Phylogeny of Halophila inferred from maximum likelihood and Bayesian analysis based on 615 bp (including gaps) of nrDNA sequences comprising ITS-1, 5.8S rDNA and ITS-2.

Nodes are presented only for those with bootstrap scores > 90% majority rule for maximum likelihood and > 90% majority probabilities for Bayesian probability values (ML/BI). Sequences are color-coded based on different sampling locations as in Fig. 1. Sample names with shading are sequences derived from molecular cloning.

Figure 4. Phylogeny of Halophila inferred from maximum likelihood and Bayesian analysis based on 440 bp of the rbcL gene.

Nodes are presented only for those with bootstrap scores > 90% majority rule for maximum likelihood and > 90% majority probabilities for Bayesian probability values (ML/BI). Sequences are color-coded based on different sampling locations as in Fig. 1.

Figure 5. Component loadings for the first of two principal components of the PCA of morphological traits with convex hull of different sample groups.

LL, lamina length; LW, lamina width; VA, crossed vein angle; DE, distance between marginal vein and lamina edge; LW/LL, lamina width/lamina length, and HLW/DE, half lamina width/distance between marginal vein and lamina edge.