Phylogenetic relationships of 'Polyalthia' in Fiji

Abstract The genus Polyalthia (Annonaceae) has undergone dramatic taxonomic changes in recent years. Nine Polyalthia species have historically been recognized in Fiji, all of which have subsequently been transferred to three different genera, viz. Goniothalamus, Huberantha and Meiogyne. The transfer of six of these species has received strong molecular phylogenetic support, although the other three species, Polyalthia amoena, P. capillata and P. loriformis [all transferred to Huberantha], have never previously been sampled in a phylogenetic study. We address this shortfall by sampling available herbarium specimens of all three species and integrating the data in a molecular phylogenetic analysis. The resultant phylogeny provides strong support for the transfer of these species to Huberantha. The taxonomic realignment of all nine Fijian species formerly classified in Polyalthia is also clearly demonstrated and supported by the resultant phylogeny. The updated taxonomic treatments of the nine species, a key to the three genera and a key to the Fijian Huberantha species are provided.

In Fiji, ten species were published under the name Polyalthia (Seemann 1861; Gillespie 1931;Smith 1936Smith , 1950Smith , 1978. In Smith's (1981) revision of Fijian Polyalthia, nine species were accepted: P. amoena A.C.Sm., P. amygdalina (A.Gray) Gillespie, P. angustifolia A.C.Sm., P. capillata A.C.Sm., P. habrotricha A.C.Sm., P. insularis (A.C.Sm.) A.C.Sm., P. laddiana A.C.Sm., P. loriformis Gillespie, and P. vitiensis Seem. The tenth species, P. pedicellata A.C.Sm., was treated as a synonym of P. vitiensis (Smith 1981). Among the nine accepted species, P. insularis was later recognized as  (Xue et al. 2014). A fifth species, Polyalthia angustifolia, was transferred to Goniothalamus as G. angustifolius (A.C.Sm.) B.Xue & R.M.K.Saunders (Tang et al. 2013); although the lack of flowers in the type specimen precluded an identification as Goniothalamus based on the connivent inner petals, the evidence from an unpublished molecular phylogeny based on sequences of the type specimen was strong enough to support the transfer (Xue 2013). The transfer was later supported in a published phylogenetic analysis with a larger taxon sampling of Goniothalamus (Tang et al. 2015). The remaining four species-P. amoena, P. capillata, P. loriformis, and P. vitiensiswere transferred to Huberantha based on a morphological study as Huberantha amoena (A.C.Sm.) Chaowasku, H. capillata (A.C.Sm.) Chaowasku, H. loriformis (Gillespie) Chaowasku, and H. vitiensis (Seem.) Chaowasku (Chaowasku et al. 2015) [initially under the generic name Hubera Chaowasku (Chaowasku et al. 2012), although this name was considered illegitimate (Chaowasku 2013;Applequist 2014)]. All nine Fijian Polyalthia species have therefore been realigned to three different genera. Turner and Utteridge (2017) recently reviewed the taxonomy and distribution of Pacific Annonaceae and incorporated the above-mentioned treatments of Fijian Polyalthia species. It is noteworthy that although the treatment of five of the Fijian Polyalthia species has been supported by molecular studies (Xue 2013;Xue et al. 2014;Tang et al. 2015), the transfer of the other four species to Huberantha was only based on morphological comparison (Chaowasku et al. 2012(Chaowasku et al. , 2015. Thomas et al. (2015) sampled Huberantha vitiensis (as 'Hubera vitiensis') in their phylogenetic study while studying the origins of intercontinental disjunctions in Annonaceae, and confirmed its taxonomic affinity with Huberantha. The other three species have never previously been sampled in a phylogenetic study.
As the genus Huberantha is taxonomically challenging and difficult to recognize, the transfer for some species based on limited collections may be problematic in the absence of molecular evidence. One example is Polyalthia floribunda Jovet-Ast from Vietnam (Jovet-Ast 1940), which was transferred to Huberantha based on its cuneate, symmetrical leaf bases, single ovule per carpel and leaf venation pattern (Turner 2016). A recent molecular phylogenetic study has revealed that the species is not congeneric with Huberantha, however, but is sister to Miliusa, although without statistical support . A new genus, Polyalthiopsis Chaowasku, was therefore erected to accommodate it . The sister relationship between Polyalthiopsis and Miliusa was later supported by Xue et al. (2020a, b) and Chaowasku et al. (2020), redefining the long-recognized sister relationship between Huberantha and Miliusa in previous studies (Mols et al. 2008;Saunders et al. 2011;Xue et al. 2011Xue et al. , 2012Chaowasku et al. 2012Chaowasku et al. , 2014.
As nomenclatural transfers based solely on morphological data can sometimes be misleading, molecular phylogenetic data can provide invaluable evidence for confirming correct taxonomic placement. To avoid such errors, we have therefore sampled the remaining three Fijian Huberantha species and undertaken a phylogenetic study to confirm their taxonomic placements.

Materials and methods
Three Fijian Huberantha species that lack DNA sequence data-H. amoena, H. capillata and H. loriformis-were sampled in this study to verify their generic position. The other six previously recognized Fijian 'Polyalthia' species were also included in this study. Sequence data for three commonly used chloroplast regions (matK, rbcL and trnL-F) were newly generated for the three Huberantha species. Sequences for other taxa were downloaded from the nucleotide database of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov). The final data matrix comprised a total of 77 Annonaceae species, representing the major clades in the family. The samples, localities and GenBank accession numbers are listed in the Appendix 1.
The phylogenetic trees were reconstructed using Bayesian Inference (BI) and maximum likelihood (ML) methods. Detailed information regarding DNA extraction, PCR amplification, and primer sequences are available , as is information on sequence alignment, model selection of the sequence matrix constructed and methods in tree reconstruction .

Results
The concatenated alignment of the 77-taxon dataset consisted of 3,659 aligned positions (trnL-F: 1,475 bp; matK: 834 bp; and rbcL: 1,350 bp). The Bayesian and ML analyses resulted in similar topologies. The 50% majority-rule consensus tree resulting from the Bayesian analysis under the three-partitioned model is shown as Fig. 1. The results are consistent with previous phylogenetic analyses of the family in which the backbone of the tribe Miliuseae remains largely unresolved.
The Fijian species previously assigned to Polyalthia are retrieved in three distinct clades (Fig. 1

Discussion
The transfer of Polyalthia amoena, P. capillata and P. loriformis to Huberantha is supported here in a molecular phylogenetic analysis for the first time. The four Fijian Huberantha species form a well-supported clade that shows a close affinity with H. nitidissima (distributed in Papua New Guinea, Australia, and New Caledonia) and H. jenkinsii (distributed in continental Asia and western Malesia). The clade comprising Polyalthia nitidissima and the Fijian 'Polyalthia' species is well separated from its sister clade consisting of species from continental Asia, Africa and Madagascar; this is consistent with the phylogeny including only one Fijian Huberantha species (H. vitiensis) published by Thomas et al. (2015).
Huberantha can be distinguished from other closely related genera by a combination of characters, including leaves with reticulate tertiary venation, axillary inflorescences, a single ovule per ovary (and therefore single-seeded monocarps), seeds with a flat to slightly raised raphe, spiniform(-flattened peg) ruminations of the endosperm, and pollen with a finely and densely granular infratectum (Chaowasku et al. 2012). It resembles Polyalthia and Polyalthiopsis in having brochidodromous foliar venation with reticulate tertiary veins. Polyalthia differs in having a generally asymmetrical leaf base and ovaries with 2-6 ovules . Polyalthiopsis differs in having foliar glands, petioles with transverse striations when dry, a leaf midrib that is raised adaxially in vivo, ovaries with 1-2 ovules, and lamelliform endosperm rumination Xue et al. 2020a). These four Fijian Huberantha species all possess a symmetrical leaf base, a flat leaf midrib adaxially, axillary inflorescences and single-seeded monocarps (Fig. 2). It is noteworthy that the monocarps are much larger, however, especially in P. capillata (25-30 cm long, 0.5-1cm broad; Fig. 2B), P. loriformis (up to 4 cm long, 1 cm broad; Fig. 2C) and P. vitiensis (up to 4.5 cm long, 1.5 cm broad; Fig. 2D) (Gillespie 1931, Smith 1950. Other Huberantha species, including H. nitidissima and H. jenkinsii, have much smaller monocarps, with the largest dimension rarely exceeding 1 cm. Seed size is possibly correlated with various factors, including overall plant size and architecture, dispersal agents, habitat and insularity (Bellot et al. 2020): the larger fruits of these Fijian Huberantha species probably reflect adaptations to alternative dispersal vectors.
The taxonomic placement of Goniothalamus angustifolius and the four Meiogyne species (M. amygdalina, M. habrotricha, M. insularis and M. laddiana) are confirmed in our study (Fig. 1): G. angustifolius is sister to another Fijian Goniothalamus species, G. monospermus; and the four Fijian Meiogyne species form a well-supported clade, although the relationship among these species is not well resolved since our analysis is based on only three chloroplast regions. The topology is consistent with a betterresolved topology based on seven markers reconstructed by Thomas et al. (2012) and Xue et al. (2014), however, with the Fijian clade sister to M. amicorum from Tonga, together forming a well-supported clade within the Australian-Pacific clade of Meiogyne species , Xue et al. 2014.

Conclusions
The transfer of Polyalthia amoena, P. capillata and P. loriformis to Huberantha is supported here in a molecular phylogenetic study for the first time. The phylogenetic analyses of previous Fijian Polyalthia species confirm that this group is a highly heterogeneous assemblage, with nine species now divided into three distantly related genera, viz. Goniothalamus, Huberantha and Meiogyne. The updated taxonomic treatments of the nine species, a key to the three genera and a key to the four Huberantha species, are provided below.   fig. 1