Molecular phylogeny of Atractocarpus (Rubiaceae): taxonomic implications for several New Caledonian Gardenieae species

Background and aims – New Caledonia is a hotspot of biodiversity in the world. Among the most diverse New Caledonian plant families is Rubiaceae, which consist of 30 genera containing 220 species, with a level of endemism of 93%. The tribe Gardenieae is represented by four genera, Gardenia (8 species), Aidia (2 species), Randia (7 species), and Atractocarpus (10 species). As Randia has now been restricted to the Neotropics, the New Caledonian Randia species remain unplaced within the tribe. Atractocarpus is a Pacific genus, easily characterized by long imbricated stipules, a feature also present in the Randia species and in several Gardenia species in New Caledonia. The aims of the present study are to test the monophyly of Atractocarpus and to assess the phylogenetic placement of the Randia and Gardenia species with long imbricated stipules within Gardenieae and specifically their relationships with taxa of the Porterandia group to which Atractocarpus belongs. Pacific Gardenieae, with a focus on the Porterandia group, in a Bayesian phylogenetic reconstruction (cpDNA: trnTF and rpl32 , and nrDNA: ITS). Key results – Our study provides a mostly supported consensus tree topology of the Porterandia group. Five Gardenia and seven Randia species fall within a clade that comprises the New Caledonian Atractocarpus species, rendering both Atractocarpus and Gardenia polyphyletic. Conclusion – We enlarge the delimitation of Atractocarpus to include 12 New Caledonian Randia and Gardenia species. New Caledonia is consequently confirmed as the centre of diversity for Atractocarpus with 31 species. According to our study, three genera of Gardenieae occur in the archipelago: Aidia , Gardenia , and Atractocarpus .


INTRODUCTION
New Caledonia is a hotspot of biodiversity isolated in the west Pacific Ocean. The exceptional flora of this archipelago mainly originated from relatively recent colonisation events from surrounding islands and the Asian and Australian continents after the emersion of the New Caledonian island group about 37 Mya (Pillon 2012). Several plant families have undergone a large diversification. Rubiaceae is among the largest New Caledonian plant families, with ca 220 species (Munzinger et al. 2020). The largest rubiaceous genus is Psychotria L. with ca 80 species (Barrabé et al. 2014), followed by Ixora L., and Cyclophyllum Hook.f with 19 species each. Like Cyclophyllum Hook.f., Atractocarpus Schltr. & K.Krause has its centre of species diversity in New Caledonia (Mouly & Jeanson 2015).
Atractocarpus is a member of the Porterandia group , together with the Asian genera Bungarimba K.M.Wong, Catunaregam Wolf (also African), Deccania Tirveng, Duperrea Pierre ex. Pit., Porterandia Ridl., Rubovietnamia Tirveng., Tamilnadia Tirveng. & Sastre, Tarennoidea Tirveng. & Sastre, Vidalasia Tirveng, plus the two genera Brachytome Hook.f. and Dioecrescis Tirveng. Atractocarpus was initially described for a single New Caledonian species (Schlechter & Krause 1908) and took its name (atraktos: a spindle, carpos: a fruit) from the fusiform berry fruit of the type species A. bracteatus Schltr. & K.Krause (Mouly 2012). Then, 13 species restricted to the New Caledonian archipelago were described in or transferred to Atractocarpus (Guillaumin & Beauvisage 1913;Guillaumin 1930) based on the shared character of numerous seeds merged within the entire placentas ( fig. 1G). Some of these do not have the fusiform fruit of the type species (e.g. spherical as fig. 1C, obpyriform as fig. 1D, or ovoid) but have fruits with a somewhat hard endocarp ( fig. 1H). These "Gardenia-like fruits" (Eriksson & Bremer 1991) are berry-like drupes, often large, with a very fibrous mesocarp and/or endocarp and a pulpy placenta when mature. Later on, Green (1990) included an Australian species from Lord Howe Island, Atractocarpus stipularis (F.Muell.) Puttock ex. P.S.Green, because of the elongated and imbricated stipules common to all species of the genus and the fruit structure. As a result, the genus Atractocarpus was no longer a New Caledonian endemic. After a phylogenetic study of the Australian and Pacific Gardenieae (Puttock 1999;Puttock & Quinn 1999) broadened the circumscription of Atractocarpus to include the genera Neofranciella Guillaumin, Sukunia A.C.Sm., Sulitia Merr., and Trukia Kaneh., plus several Australian Randia and Gardenia species. From Puttock's circumscription of the genus Atractocarpus, the newly accepted diagnostic features were elongated imbricated stipules lacking yellow wax ( fig. 1B; a product secreted by colleters on the inside of the stipules), unisexual flowers (individuals dioecious or gynodioecious), and fruits with a woody endocarp. Following Puttock's (1999) circumscription, Atractocarpus consists of 29 species.
Recently, when describing the genus Bungarimba K.M. Wong, Wong (2004) questioned the monophyly of Atractocarpus based on morphological comparison of taxa and phylogenetic data. Indeed, this author showed that Atractocarpus sensu Puttock was paraphyletic, with Porterandia nested within it (Wong 2004: fig. 2), and Bungarimba distinct from the Atractocarpus-Porterandia clade, though without branch support. The results of  also questioned the circumscription of Atractocarpus proposed by Puttock (1999), showing a close relationship of the three genera Sukunia, Trukia, and Bungarimba to Catunaregam and Deccania, rather than to Atractocarpus and Neofranciella. However, very few of the internal Porterandia group relationships received high posterior probabilities in , meaning that it was preferable to consider the relationships unresolved. More recently, Kainulainen et al. (2017) included one species of Atractocarpus and one of Sukunia in a biogeographical study of Indian Ocean Rubiaceae. These two representatives were not closely related, but no support for branching was provided. Therefore, there is no strong evidence so far that Puttock's concept of Atractocarpus is not reliable.
The New Caledonian Randia and Gardenia species were not studied by Puttock (1999), and their phylogenetic positions in Gardenieae remain problematic. Indeed, Randia in its old circumscription was shown to be polyphyletic (Persson 2000;Andreasen & Bremer 2000) and is now restricted to the Neotropics (Gustafsson & Persson 2002), leaving several New Caledonian Randia species unplaced within the Gardenieae. Within the New Caledonian Gardenia, only Gardenia aubryi Vieill., G. oudiepe Vieill., and G. urvillei Montrouz. have stipules covered by yellow wax, a typical feature in Gardenia. Among the other New Caledonian Gardenia species, at least one has a monocaulous habit and is dioecious (Gardenia conferta Guillaumin), two common features in Atractocarpus (Puttock 1999) but not in Gardenia, and another one has elongated fruits (Gardenia colnettiana Guillaumin), which are more common in Atractocarpus than in Gardenia. On the other hand, G. conferta differs from Atractocarpus sensu Puttock (1999) by corolla lobes shorter than the corolla tube. Kainulainen et al. (2017) included seven of the New Caledonian Gardenia; they found that the three species with stipules covered by yellow wax were embedded within the Gardenia group, whereas the four other species (e.g. G. conferta and G. colnettiana) were associated with Atractocarpus heterophyllus (Montrouz.) Guillaumin & Beauvis. These findings clearly show that the taxonomic position of the New Caledonian Randia and Gardenia need to be investigated thoroughly.
As New Caledonian Gardenieae species relationships are still problematic, we decided to dedicate a study in order to obtain a well-defined molecular phylogenetic structure for relationships within the Porterandia group, with a focus on Atractocarpus and New Caledonian Gardenieae. The present study aims at 1) testing the monophyly of the genus Atractocarpus as delimited by Puttock; 2) assessing the phylogenetic positions and relationships of the New Caledonian Atractocarpus, Gardenia, and Randia; and 3) providing taxonomic treatments for supported relationships, when necessary.

Material
To cover the diversity in Gardenieae as much as possible, and thus be able to place New Caledonian species with unknown affinities more accurately, we sampled 13 genera and 63 species from both silica and herbarium material (K, NOU, P, UPS, S; acronyms follow the Index Herbariorum; Thiers continuously updated). We were, however, unable to obtain material or sequences for Sulitia species from the Philippines.
The taxa represent a quite exhaustive sampling of New Caledonian Gardenieae, including 11 undescribed new species (Atractocarpus sp. 2 to A. sp. 12), with a special emphasis on Atractocarpus, Randia, and Gardenia, plus two New Caledonian Aidia species. We included several available Pacific and South-East Asian Gardenieae species (including another undescribed species of Atractocarpus from Tahiti; A. sp. 1) and other Gardenieae representatives from the different groups recognized by . Taxa of the Octotropideae s.l. ) clade were selected to root the tree. The list of sequenced material is available as supplementary file 1. The 12 undescribed species cited here will be published in a complete revision of the genus Atractocarpus for New Caledonia by the first author of the present publication.

Laboratory procedures
We utilized information from two chloroplast regions (trnTF and rps32) and a nuclear ribosomal region (ITS). DNA was extracted, amplified, and sequenced using standard procedures as outlined in Kårehed & Bremer (2007) and Rydin et al. (2008).

Alignment and phylogenetic reconstruction
Sequences were aligned using the software Se-Al v.2.0 (Rambaut 1996). Insertion/deletion events were inferred by eye. Gaps were treated as missing data in the alignment and were added as binominal characters (absent or present) at the end of the matrix.
Bayesian analyses were performed using MrBayes v.3.1.2 (Huelsenbeck & Ronquist 2001;Ronquist & Huelsenbeck 2003). For each single gene data set, the best performing evolutionary model was identified under three different model selection criteria: Akaike information criterion (AIC) (Akaike 1973), AICc (a second order AIC, necessary for small samples), and the Bayesian information criterion (BIC) (Schwartz 1978). We performed these calculations using MrModeltest v.2.4 (Nylander 2004). Ambiguous alignments and insertions-deletions were excluded from the data sets for analyses. For single gene analyses, the best performing model under the AICc criterion was selected, 10 7 generations were run, with a sample frequency of 1000 and four parallel chains. A flat dirichlet prior probability (all values are 1.0) was selected for the substitution rates (revmatpr) and the nucleotide frequencies (statefreqpr). The prior probability for the shape parameter of the gamma distribution of rate variation (shapepr) was uniformly distributed over the interval (0.1, 50.0). For analyses using a gamma distribution with a proportion of invariable sites, we specified a prior probability for this proportion (pinvarpr), uniformly distributed over the interval (0.0, 1.0). For the combined analyses, model selection and settings were selected in the same way as for single gene analyses, in order to ensure that the analyses represented an adequate sample of the posterior distribution, five million generations were run. Partitions were unlinked so that each partition was allowed to have its own set of parameters.

RESULTS
Separate analyses of all markers resulted in more or less resolved tree topologies, with low to high support for clades. Relationships of the main clades highlighted on the tree ( fig. 2) were often not resolved or if so, poorly supported. However, these main clades were retrieved in most of the analyses. The combined data from the three markers resulted in a much more resolved topology, where the main clades were well-supported and their relationships received strong support ( fig. 2).
In the combined phylogeny, the ingroup forming the Gardenieae was monophyletic (PP = 1.00). The earliest divergent lineage consisted of Gardenia species (PP = 1.00) and included the type species of the genus, G. jasminoides J.Ellis, and the New Caledonian species G. aubryi, G. oudiepe, and G. urvillei. They represented the Gardenia group sensu . The second lineage represented the Randia group sensu  and comprised two specimens of Randia aculeata L. Within the ingroup, the genera Aidia, Atractocarpus, Gardenia, and Randia were not monophyletic when including the New Caledonian native species. First, the two New Caledonian native Aidia species formed a clade with the genera Tamilnadia, Brachytome, and Dioecrescis (not supported; PP = 0.60), but not with the other Aidia species from Africa and Asia.
Within the lineage that included most of the Atractocarpus species (hereafter called Atractocarpus s.l.; fig. 2

DISCUSSION
When redefining Atractocarpus, a century after its original description, Puttock (1999) and Puttock & Quinn (1999) did not extensively treat the New Caledonian species. The inclusion in our phylogenetic study of a large number of species of Atractocarpus and several uncertainly placed New Caledonian Randia and Gardenia demonstrates the paraphyly of Atractocarpus sensu Puttock and the need for a new circumscription of New Caledonian Gardenieae species associated to it in the phylogeny.

Paraphyly of Atractocarpus sensu Puttock and consequences
Most of the Gardenieae species restricted to New Caledonia grouped with the New Caledonian Atractocarpus species in a NC clade sister to a Lord Howe Island and Australian Atractocarpus clade. Several of these species bear an elongated pendulous fruit, the initial characteristic feature for the genus Atractocarpus. Most of the Randia and Gardenia species here placed within the NC clade have unisexual flowers, common in Atractocarpus. All have elongated imbricated stipules lacking yellow wax that constitute a diagnostic character of Atractocarpus (Guillaumin 1930;Puttock 1999;Mouly 2012).
To recognize only monophyletic lineages, the wellsupported tree topology forces us to split Atractocarpus sensu Puttock or to lump the New Caledonian Randia and four Gardenia within it. The internal position of the type species A. bracteatus and the relative homogeneity of the representatives included in the lineage, with an available diagnostic feature for the lineage (linear imbricated stipules), support the recognition of a broad concept for the genus Atractocarpus (Atractocarpus s.l.; fig. 2). This necessitates maintaining the name Atractocarpus, the oldest available name, and extending the generic limits to include 12 additional species and 12 novelties. The species newly included under Atractocarpus generate few changes in its previous circumscription (see Puttock 1999), such as the corolla lobes possibly shorter than the tube (e.g. in Gardenia conferta) and the presence of monoecy or hermaphroditism (e.g. in Randia pseudoterminalis Guillaumin).
A broad circumscription of Atractocarpus also has the advantage of maintaining the concept of Atractocarpus provided by Puttock (1999), including the previously separated genera Neofranciella, Trukia, and Sukunia. Indeed, our new phylogenetic study, using other molecular markers than previous analyses (e.g. Kainulainen et al. 2017) ended in a significantly structured and supported tree topology for Atractocarpus and its relatives. Here, we find no support for a closer relationship of Sukunia (represented by A. pentagonioides on fig. 2) to Bungarimba (as in Kainulainen et al. 2017) or of Sukunia and Trukia (represented by A. carolinensis on fig. 2) to Bungarimba (as in . To split Atractocarpus and to delimit several narrow genera makes no sense, as the internal morphological variability of Atractocarpus s.l. subclades (clades NC1, NC2 and Australian Atractocarpus; fig. 2) is almost similar to the morphological diversity between the subclades. Plus, we have been unable to find morphological synapomorphies to diagnose the different subclades within Atractocarpus s.l. (fig. 2). Consequently, we favour the recognition of the lineage called Atractocarpus s.l. (fig. 2) enlarged to include several New Caledonian Randia and Gardenia species.
The tree topology for Atractocarpus s.l. is congruent with the geographical distribution and may be a frame for an infra-generic classification. However, the present sampling of species from outside New Caledonia is not sufficient to give a comprehensive view of the morphological variability within the genus. A possible infra-generic delimitation of Atractocarpus should be addressed in a further study, due to missing species from Australia, Fiji, and Papua-New Guinea in our phylogenetic sampling.

Emended generic description of Atractocarpus s.l.
Accepting a broad concept of Atractocarpus, in order to render the taxonomic concept monophyletic, results in a well-diversified lineage with 53 species in the genus (39 included in our phylogeny) and 31 species indigenous to New Caledonia when including the undescribed novelties. Atractocarpus becomes the second most species-diverse genus of Rubiaceae in New Caledonia (Munzinger et al. 2020) and one of the most species-rich genera in the tribe Gardenieae in general . New Caledonia remains the centre of diversity for Atractocarpus, with ca 60% of the species. Following our results, an emended description of Atractocarpus is provided and 12 New Caledonian Randia and Gardenia species are transferred to it.
With regard to Puttock's delimitation (1999) of Atractocarpus, the main emended characteristics are the sexual system with inclusion of monoecy with hermaphroditic flowers, monocaulous treelets (see Bruy et al. 2018), the corolla lobes shorter than the corolla tube, and the elongated thin cylindrical fruits with fleshy and soft endocarp. Accordingly, there is no clear diagnostic character for the genus within the Porterandia group, but the combination of the elongated stipules imbricated in early stages (even fused; fig. 1B) and the corolla tube urceolate (short to elongated) with a constriction at the mouth (fig .  1E) seems sufficient to place a species within the genus as circumscribed here.

The tribe Gardenieae in New Caledonia
According to the database FLORICAL (Morat et al. 2012;Munzinger et al. 2020), the family Rubiaceae is represented by 220 species in New Caledonia, to which we can add the 12 undescribed Atractocarpus species. The tribe Gardenieae, with 31 Atractocarpus species (30 endemic + one native), two endemic Aidia species, and three endemic Gardenia species, covers 15.5% of the native Rubiaceae diversity of the New Caledonian islands, with 97% of endemism. Gardenia species are largely distributed on the main island; G. urvillei occurs in lowland sclerophyll forests or rarely bushes, G. oudiepe in forested vegetation from sea level to mountain, and G. aubryi is common in bushes on ultramafic substrates. The Aidia species, A. congesta and A. vieillardii, are dioecious, presenting sexual dimorphism. They mostly occur in forested environments on the internal mountain chains of the main island, and are both endemic to New Caledonia. These two Aidia species do not fall together with the two other Aidia species from Asia and Africa included in our study, but appear included within the Porterandia group (sensu ). According to this, a specific investigation should be initiated to assess the observed polyphyly of Aidia with an appropriate species and geographic sampling. Atractocarpus in New Caledonia is adapted to a broad ecological range (Bruy et al. 2018) and occurs in most of the vegetation types, from sea level to the summit of the highest mountain. Atractocarpus sezitat, described from New Caledonia, is the sole species of the genus that reaches the Loyalty Islands as well as Vanuatu. All other New Caledonian Atractocarpus species are restricted to the archipelago, and several of them are micro-endemic to very narrow areas or specific environments, such as A. pancherianus in sclerophyll forests or A. colnettianus on the north-eastern mountains. A revision of the genus for New Caledonia is under progress by the first author of the present study.

CONCLUSION
The present study contributes to a better understanding of the genus Atractocarpus, based on studies initiated by Puttock & Quinn (1999) and Puttock (1999). The present molecular phylogenetic study allows the establishment of a new circumscription of the genus, with the integration of 12 previously misplaced Randia and Gardenia species from New Caledonia. Under this new circumscription, the New Caledonian Atractocarpus lineage is monophyletic, consisting of 20 described species (12 transferred to the genus in the present study), plus 11 species in need of description. Atractocarpus as a whole contains 53 species from the west to central Pacific Islands and Australia, including 11 novelties from New Caledonia and one from French Polynesia. Gardenieae are finally represented by three genera in New Caledonia: Aidia with two native species, Atractocarpus, and Gardenia, both with three native species.