Molecular Phylogeny of the Philippine Endemic Genus Antherostele Bremek . ( Rubiaceae ) Inferred from ITS Data ( nrDNA ) and Its Conservation Status

Antherostele Bremek. is an understudied genus of Rubiaceae endemic to the Philippines formerly comprising four species of shrubs or small trees distributed in Luzon and Visayas. In this first molecular study of the genus, the ITS (nrDNA) was utilized to determine the monophyly of Antherostele, reconstruct its phylogeny, and resolve its placement in Rubiaceae. Furthermore, an update on the assessment of its conservation status of the included species in the genus was done. Antherostele is monophyletic and is nested within the tribe Urophylleae of subfamily Rubioideae which confirms its segregation from Urophyllum and its placement within Urophylleae as proposed by earlier authors based on morphology. However, the relationships of Urophylleae members remain unresolved. Antherostele is united by reflexed corolla, hairs on upper side of the corolla lobes and syngenesious stamens. The montane A. luzoniensis with small leaves is found to be sister to the rest of Antherostele species. Antherostele is hypothesized to originate from montane regions and diversified by forming larger leaves following dispersal to the shaded forest understory. From the five species of Antherostele currently recognized in the study, two are endangered (A. banahaensis, A. grandistipula) and three are critically endangered including a new species (A. callophylla, A. luzoniensis, A. sp. nov.) following the criteria of IUCN.


Introduction
Rubiaceae (coffee family) is the fourth largest family of angiosperms in the world with about 660 genera and 13,000 species [1].It is predominantly woody and well represented in the tropics, usually comprising the understory of the forest.In the Philippines, Rubiaceae contains the largest number of indigenous species among families of dicotyledonous flowering plants.It is represented by about 80 genera and 550 species, which comprise 13% of the global Rubiaceae genera [2].Despite this importance, Rubiaceae remains understudied and incompletely known especially in the tropics where most of the endemics are found [1].In the Philippines, more than 15 genera contain endemic species [3] and at least four of these genera [Antherostele Bremek., Greeniopsis Merr., Sulitia (Merr.)Ridsdale, Villaria Rolfe] are endemic [2,4,5].
Antherostele is a widely accepted distinct genus segregated from Urophyllum Wall.by Bremekamp [6] based on morphological features such as long pedicels, domatia on the underside of leaves, corolla lobes distinctively hairy above, and fused anthers.Bremekamp [6] recognized four species of Antherostele, namely A. banahaensis (Elmer) Bremek., A. callophylla Bremek., A. grandistipula (Merr.)Bremek.and A. luzoniensis (Merr.)Bremek.Except for A. callophylla, three of the species were originally described as Urophyllum.However, the descriptions of the genus and its species provided are based solely on limited herbarium material; hence, some information, mostly on reproductive parts, are lacking.The genus has not been updated and revised since Bremekamp [6].Recent collections of this endemic genus are wanting because herbarium specimens from both local herbaria, e.g.College of Agriculture Herbarium-University of the Philippines (CAHUP), Philippine National Herbarium (PNH), Philippine University Herbarium (PUH); and foreign herbaria e.g., Herbarium of the Arnold Arboretum (A), Conservatoire et Jardin botaniques de la Ville de Genève Herbarium (G), Nationaal Herbarium Nederland (L), New York Botanical Garden Herbarium (NY), and United States National Herbarium (US), are scarce.Furthermore, the endemic genus Antherostele and its current placement in Rubiaceae have not been confronted by any molecular study.Its establishment as a genus separated from Urophyllum [6] and present inclusion in Urophylleae under Cinchonoideae [1,7] were based on morphology alone.
The internal transcribed spacer (ITS) is part of the transcriptional unit of the nuclear ribosomal DNA (nrDNA) [8] which consists of a repeated unit that exists as tandem repeats [9] in the plant genome.It is one of the most extensively used genetic markers in phylogenetic studies in angiosperms [8][9][10] and has been proven to be of utility for several reasons.The ITS is relatively small (ca.700 base pairs) and is flanked by highly conserved genes (18S rRNA and 26S rRNA) which promotes ease in amplifying and sequencing [8].ITS exists also as thousands of copies in most plant genomes thus making it very easy to detect [9].These copies undergo concerted evolution through unequal crossing over and gene conversion, thereby homogenizing all the copies within the genome [8].Moreover, forty percent of the ITS are highly conserved across all angiosperms sequenced, which accounts for its high sequence alignability.ITS is also rapidly evolving and is very useful in resolving issues in the generic and specific levels [9].Lastly, the ITS spacers (ITS 1 and 2) have presumptive role in the maturation of nuclear ribosomal RNAs that accounts for its subjection to evolutionary constraints [8].In Rubiaceae, several works have attested to the utility of ITS data in resolving taxonomic relationships [e.g., [11][12][13][14][15].In the current study, the ITS is utilized in addressing taxonomic issues in Antherostele.
In 2007, the Department of Environment and Natural Resource released DENR Administrative Order No. 2007-01 [16] establishing a national list of threatened Philippine Plants and their categories.This list included all known species of Antherostele and categorized them as vulnerable.With the present study, this classification could be updated since knowledge about Antherostele is still inadequate especially its status in the wild.
With the lack of understanding of Antherostele and need for further assessment of its conservation status, the present study aims to test the monophyly of Antherostele, reconstruct the phylogeny of the genus and assess phylogenetic relationships of closely related genera within their resolved tribal position using the internal transcribed spacers (ITS) of the nuclear DNA.It also intends to reevaluate the status of Antherostele species in the wild following IUCN red list categories and criteria [17].The present study is the first molecular work on Antherostele with an update on its conservation status.
Collection was done by obtaining the flowering and/or fruiting branch of the specimen.At least three branches were obtained and tagged from each of at least three individuals for every species for preservation as herbarium specimens.Each branch was inserted in a folded newspaper and placed in a polyethylene bag.Denatured alcohol was poured into the bag to preserve the plant until they were brought into the laboratory for processing.During collection, morphological features, especially ephemeral characteristics, i.e., odor, color etc., were noted.Some fruits and flowers were pickled in ethyl alcohol (1:1) for detailed studies in the laboratory.Two to three fresh leaves from each species were obtained and were placed in a 5" × 4" re-sealable polyethylene bag containing silica gel for molecular study [18].
The amplified PCR products were then resolved in a 1% agarose gel from 5 μl of the reaction mix using 1× TAE buffer [21] for verification and identification of PCR products.Subsequently the PCR products were purified using the Qia-Quick Purification kit (Qiagen) to remove excess template, primer-dimers, and oligonucleotides.The purified DNA samples, along with the primers and the documented gel, were submitted to the Department of Plant Systematics, Bayreuth University, Germany for sequencing.

DNA Sequence Analysis
Six ITS sequences of Antherostele are newly reported here.Their GenBank accession numbers and voucher information are listed in Table 2. Aside from the Antherostele ITS data, 52 ITS sequences from Luculia grandifolia Ghose, L. gratissima (Wall.)Sweet, and several accessions under Rubioideae including Colletoecema dewevrei (De Wild.)E.M.A. Petit and representatives from Urophylleae and Ophiorrhizeae [22,23] were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and added in the analysis, giving a total of 58 ITS sequences.The genus Luculia, which has been shown to be the earliest diverging lineage in Rubiaceae [24], was used as an outgroup to root the ITS tree.The ITS sequences were edited using Bioedit v.7.0.4.1 then alignment of the sequences was performed using MacClade v. 4.0.The boundaries of the coding and spacer regions were deter- mined using previously published ITS sequence data [19,25].Excluding uninformative characters, a maximum parsimony analysis of ITS data was performed with PAUP* v. 4.0 [26] using heuristic search, with the MULTREES option on, tree-bisection-reconnection branch (TBR) swapping, swap on best only in effect, and 10,000 random addition sequences.Characters were treated unordered and weighed equally and gaps were treated as missing data.A strict consensus tree was computed from all the parsimonious trees generated by the heuristic search.The consistency index [CI; 27] and retention index [RI; 28] were calculated to estimate homoplasy.Bootstrap (BS) analysis [29] was also performed to determine robustness of the clades formed using 10,000 pseudoreplicates, with the MULTREES option off, TBR swapping, and five random addition sequences.The resulting bootstrap values were transferred to the supported clades in the strict consensus tree.Trees produced from parsimony analysis were viewed in TreeView v.1.6.6 [30] and were edited in Mesquite v.2.74 [31] and CorelDRAW X3.The following criteria were followed on describing the result of the bootstrap analysis: clades receiving a bootstrap support of 50% -69%: weakly supported; 70% -85%: moderately supported; and 86% -100%: strongly supported [19].

Conservation Status Assessment
The status in the wild of the species of Antherostele was evaluated using applicable criteria provided by IUCN version 3.1 [17].Estimation of subpopulation involved direct observation of the individual species in the study site.The area of occupancy was based on the information provided by herbarium collections (A, CAHUP, G, HUH, K, L, NY, PNH, PUH, US).Current threats and imminent dangers to the survival species were also noted.Furthermore, the quality of the habitat was also generally assessed.

DNA Sequence Characteristics
The length of ITS sequences from all taxa varied from
Antherostele is monophyletic and forms a well-resolved clade (BS = 100%).This supports the segregation of Antherostele from Urophyllum as proposed by Bremekamp [6].Antherostele is characterized by its reflexed corolla, hairs on the corolla lobes and united anthers (Figure 2).The present analysis also confirms the placement of Antherostele in Urophylleae (BS = 74%) which corroborates Robbrecht [1].The present study is the first mo- lecular work that confirms monophyly of Antherostele and its placement within the said tribe.However, the sister group of Antherostele within Urophylleae cannot be determined in the current study as relationships within Urophylleae are not completely resolved using the present data (Figure 2).

Phylogeny of Antherostele
The Antherostele clade is well-resolved and shows A. luzoniensis as sister to the rest of the Antherostele (Figure 2).Antherostele luzoniensis is separated from the rest by thriving in cool montane forests located in high elevations (1500 -1700 masl) and has relatively smaller leaves as opposed to the remaining Antherostele species which prefers humid forest at low to medium elevation (300 -1000 masl) and has larger leaves.It is hypothesized that Antherostele started as a montane species and diversified following dispersal down the shaded forest understory.
The current study estimates that from its small leaves, Antherostele started adapting by forming larger leaves to compensate for the reduced illumination below the forest canopy.Furthermore, ITS data suggests that Antherostele sp.nov.(Obico and Alejandro, submitted) is the sister group of the remaining understory species followed by A. grandistipula.Antherostele sp.nov. is notable for it is found within the geographic range of A. grandistipula, particularly in Samar.There was no evidence from morphology that these two species form natural hybrids, as both are distinct from each other.This morphological distinction of both species, while being found in similar locality, may be attributed to sympatric speciation probably caused by niche differentiation.Antherostele grandistipula seems to prefer areas along streams while Antherostele sp.nov.on the interior of the forest.However, evidences at hand appear insufficient as additional collections and field observations of A. sp.nov.are needed to verify this statement.Antherostele sp.nov.seems to be rare in the collection site.Antherostele banahaensis (Figure 3) and A. callophylla form a pair with a strong support (BS = 83%).This indicates a close relationship between them.The two species are almost similar morphologically but can somehow be distinguished initially by longitudinal slits on branches, leaves, stipules, puberulence and other small flower details.However, these differences tend to overlap or not consistent in all specimens studied which may be due to differences in local environmental conditions and are thus not helpful distinguishing characters.A question on their distinction which could lead to their possible conspecificity could be raised.A need for revision of the genus and additional molecular study are recommended to address this concern.

Conservation Status of Antherostele Species
Based on extensive fieldwork and examination of several herbarium specimens, an assessment of the status in the wild of the species of Antherostele was done following the criteria of IUCN [17].This is an update from the present status of Antherostele based on DENR DAO 2007-01 [16]. 1) Antherostele banahaensis (Elmer) Bremek.:This species is a Luzon endemic and is found in Cagayan, Isabela, Rizal, Laguna, Quezon, and Sorsogon provinces.It prefers low to medium altitude (300 -830 masl),and habituates dipterocarp and mossy forest along gullies, creeks or streams with clay-loam soils.IUCN Red List Category: Endangered (EN; C2ai)-Population size estimated to number fewer than 2500 mature individuals.Continuing decline is projected and no subpopulation is estimated to contain more than 250 mature plants.All habitats of A. banahaensis are threatened by anthropogenic activities.
2) Antherostele callophylla Bremek.:This is another Luzon endemic and is known only from one locality, Mt.Palemlem in Ilocos Norte.It prefers medium altitude (500 -700 masl) in dipterocarp forest along the stream with clay-loam soils.IUCN Red List Category: Critically Endangered (CR; B2ab)-the area of occupancy is estimated to be less than 10 km 2 , and is known to exist at only a single location.Continuing decline is projected in the area of occupancy and quality of habitat as logging activities by the indigents in the habitat of this species are beginning to take place.
3) Antherostele grandistipula (Merr.)Bremek.:This species is found in the eastern side (Camarines Sur, Catanduanes, Samar, Leyte) of the Philippine Archipelago except for one specimen in Mindoro.This species prefers river or creek bank in forest around 175 -765 m asl with clay-loam soils.IUCN Red List Category: Endangered (EN; C2ai)-population size is estimated to number fewer than 2500 mature individuals.A continuing decline is projected and no subpopulation is estimated to contain more than 250 mature or even more than 50.Most areas occupied by A. grandistipula are fragmented forests caused by logging or mining activities before.
4) Antherostele luzoniensis (Merr.)Bremek.This species is endemic to Luzon and can be found in the provinces of Isabela, Nueva Ecija, and Aurora.It prefers montane forest along the ridge or slopes at around 1554 -1657 masl with clay-loam soils.IUCN Red List Category: Critically Endangered (CR; C2ai)-population size is estimated to number fewer than 2500 mature individuals and a continuing decline in the number of mature individuals is projected and no subpopulation is estimated to contain more than 250 mature individuals.
5) Antherostele sp.nov.This new species is found in the forest understory of Samar and Mindoro at around 100 -300 masl, near river banks or steep valley slopes with clay-loam soils.The species appears to be rare in the wild.IUCN Red List Category: Critically Endangered (CR; C2ai)-population size is estimated to number fewer than 2500 mature individuals and a continuing decline in the number of mature individuals is projected and no subpopulation is estimated to contain more than 250 mature individuals.The habitat of this species is threatened by human land use.Antherostele can only be found in the Philippines and all the included species are threatened in the wild.Moreover, three species of Antherostele (A. callophylla, A. luzoniensis and A. sp.nov.) have restricted distribution and can be considered rare based on field observation.Aggravating further the situation is the dioecy of these species which could be a disadvantage for them.The depletion or complete lost of one member of the sex due to habitat loss could lower the species reproductive capability thus reducing progenies in the future generations.All of the aforementioned obviously connote an imperative call on protecting these species.Creation of sustainable conservation efforts for all species should be taken seriously.

Future Studies
To date, there has never been any attempt to study the presence of important compounds on Antherostele, as this genus has been poorly known and its endemic range limits its accessibility.Ethnobotanical accounts on Antherostele are also lacking.Systematics has a predictive value especially with regard to the discovery of bioactive or industrially important compounds on related taxa.Based on its present placement in Rubiaceae, subfamily Rubioideae [22], Antherostele can be a potential source of anthraquinones, an organic compound which has many industrial and medicinal applications.
Furthermore, Jansen et al. [32] reported aluminum accumulation in Antherostele.Being a strong aluminum accumulator, Antherostele is a promising plant for rehabilitating soils with high levels of aluminum which is one of the main players in limiting crop productivity on acidic soils.This aspect could be explored more thoroughly in future undertakings.

Figure 1 .
Figure 1.Map showing collection sites of Antherostele species.

Figure 2 .Figure 3 .
Figure 2. Strict consensus tree derived from 2023 equally parsimonious trees based on parsimony analysis of ITS sequence data.Numbers above nodes are bootstrap support values more than 50%.The letters at the nodes refer to clades discussed in the text.OG refers to outgroup taxa.Clades are indicated by their respective bracket labels.Antherostele species are highlighted.

Table 1 . Total number of Antherostele herbarium specimens used in the study.
Molecular Phylogeny of the Philippine Endemic Genus Antherostele Bremek.(Rubiaceae) Inferred from ITS Data (nrDNA) and Its Conservation Status 1401

Table 2 . Voucher information of Antherostele materials used in this study.
*University of Santo Tomas Herbarium.