Species composition in the Balanophora laxiflora species complex (Balanophoraceae): molecular and morphological evidence

The Balanophora laxiflora species complex is one of the most taxonomically difficult lineages within the holoparasitic genus Balanophora . Over the last century, a number of contrasting opinions have been expressed regarding the identities and relationships of the species currently considered as members of this complex. Although the complex is widely distributed across tropical and subtropical Asia, previous studies have focused on its Japanese and Chinese representatives, whereas populations from Southeast Asia have received little attention. Here, we fill this gap through an integrative study with employment of a set of original specimens from several provinces of Vietnam. We present the results of a comprehensive morphological assessment that includes examination of living plants in situ and analysis of alcohol-fixed specimens using light and electron microscopy. We also provide an expanded molecular phylogenetic reconstruction of the species complex and establish the phylogenetic placement of our Vietnamese specimens. We report extensive contradictions between the characteristics of the studied specimens and the presumed morphological boundaries of the currently recognized species. In particular, the dimensions of the claviform bodies of the female inflorescence and their cuticle morphology frequently showed variants attributed to different species within a single specimen. Even when these features were excluded from consideration, 10 of the 16 studied specimens could not be assigned to any species, because each of these specimens demonstrated a mixture of character states considered diagnostic for different species, most of such plants being morphologically intermediate between B. laxiflora and B. yakushimensis . The phylogenetic results do not contradict the idea of distinctness of B. appressa , B. japonica and B. nipponica . At the same time, B. laxiflora , B. parajaponica and B. yakushimensis were inferred as non-monophyletic, and their monophyly cannot be achieved even if the morphologically intermediate specimens are conventionally assigned to species according to their phylogenetic placement. The morphological and phylogenetic evidence therefore unequivocally indicates the need for re-shaping the interspecific boundaries, with one of the possible decisions being merging of B. laxiflora , B. parajaponica and B. yakushimensis into a single species. Further analysis has shown, however, that even after such a modification, the species in the B. laxiflora complex would only weakly be differentiated from each other morphologically. Thus, recognition of the entire lineage as a single species might appear the most appropriate taxonomic solution. We highlight the importance of comprehensive sampling in studies of intricate lineages similar to the B. laxiflora species complex, especially broad geographical coverage and careful assessment of structural diversity within each analyzed population and specimen.