INTRODUCTION
The red algal order Peyssonneliales (Krayesky et al. 2009) occurs circumglobally from shallow reef flats to depths greater than 250 m (Littler et al. 1985, Ballantine and Ruíz 2011). Some members of the Peyssonneliales have been prominent elements of reefs, limestone substrates, and rhodolith beds since the Late Mississippian epoch (Wray et al. 1975, Wray 1977) or the Early Cretaceous period (Massieux and Denizot 1964, Johnson and Kaska 1965), and they remain abundant and ecologically significant on reefs today (Dethier et al. 1991, Ballantine et al. 2014, Nash et al. 2015). The order has garnered further interest due to the rising threat of spatially aggressive peyssonnelioid algal crusts (PAC), which have become more prominent on tropical reefs over the last decades and can overgrow corals, deter coral settlement, and may be less impacted by ocean acidification (Dutra et al. 2016, Edmunds et al. 2019, 2023, Stockton and Edmunds 2021). The order has received a significant increase in systematic interest in recent years, including the reassessment, description, or recognition of several genera (e.g., Zhang and Zhou 1981, Kato et al. 2006, Pestana et al. 2021) and species (e.g., Dixon and Saunders 2013, Sherwood et al. 2020, Mills and Schils 2021, Pestana et al. 2021).
Currently, 15 genera are recognized within the Peyssonneliales (Guiry and Guiry 2025). Among them, Incendia K. R. Dixon (2013, p. 84) is distinguished by secondary pit connections in the perithallus, multicellular rhizoids, and its recovery as a distinct, well-supported monophyletic clade (Dixon and Saunders 2013). The presence of secondary pit connections and enlarged cells embedded in the perithallus near the dorsal surface are vegetative features shared between most Ramicrusta D. R. Zhang and J. H. Zhou (1981, p. 538) and Incendia species. These enlarged cells are typically noticeably larger than neighboring filaments (Dixon and Saunders 2013). While initially referred to as ‘heterocysts’ (Zhang and Zhou 1981), Pueschel and Saunders (2009) hypothesized that these structures were the persistent bases of shed hairs and were therefore referred to as hairs, hair cells, or trichocytes when describing new Ramicrusta and Incendia species (e.g., Dixon and Saunders 2013, Ballantine et al. 2016, Pestana et al. 2020, Mills and Schils 2021, Sherwood et al. 2021). However, recent investigations of peyssonnelioid diversity and systematics have increasingly identified similar vegetative anatomies both within and between genera (Dixon and Saunders 2013, Pestana et al. 2021). As a result, DNA sequence data and phylogenetic analyses have become fundamental in delineating species boundaries within the Peyssonneliales (e.g., Pestana et al. 2020, 2021, Sherwood et al. 2020). The 5′ end of the mitochondrial cytochrome c oxidase subunit 1 gene (COI-5P), the primary barcode marker for red algae (Saunders and McDevit 2012), has been the principal marker used to delineate most species of Incendia and Ramicrusta (Dixon and Saunders 2013, Dixon 2018, Mills and Schils 2021, Sherwood et al. 2021). The plastid ribulose-1,5-biphosphate carboxylase large subunit gene (rbcL) has also occasionally been employed as a secondary marker when describing new Incendia species (Pestana et al. 2020).
The distributions of nearly all currently recognized Incendia species are limited to the regions from which they are described, with the exception of I. undulata K. R.Dixon, which occurs in Vanuatu, the Philippines,and western Australia (Dixon and Saunders 2013, Dixon 2018). Eight of the ten species occur in the westernPacific and Indian Oceans. The six western Pacificspecies, including I. basilii K. R. Dixon, I. crenata K. R.Dixon, I. cryptotricha K. R. Dixon, I. glabra K. R. Dixon,I.regularis K. R. Dixon, and I. undulata were describedfrom Vanuatu (Dixon and Saunders 2013). In additionto I. undulata, the other two Indian Ocean species,I.cryptica K. R. Dixon and I. homosorora K. R. Dixon,were described from western Australia (Dixon 2018).The only Incendia species found outside of the westernPacific and Indian Oceans are the two most recentlydescribed members of the genus, I. yoneshigueana Pestana, G. N. Santos, V. Cassano and J. M. C. Nunes andI.lisianskiensis A. R. Sherwood, which were describedfrom Brazil and Hawaii, respectively (Pestana et al. 2020, Sherwood et al. 2021).
Despite being a relatively small, remote tropical island in the northwestern Pacific with a total shoreline length of 244 km and a total nearshore reef area of approximately 108 km2 (Burdick 2005, Burdick et al. 2008), Guam features one of the most diverse and best documented crustose calcifying red algal (CCRA; i.e., non-geniculate calcified Corallinophycidae andcalcified Peyssonneliales) floras in the world (Mills etal. 2022). Further research into the diversity of CCRAin Guam and Micronesia remains ongoing. Over thecourse of these investigations, we collected 18 peyssonnelioid specimens belonging to the genus Incendia.Below, we describe eight new species of Incendia fromMicronesia using comparative molecular analyses andmorphological characterizations, as well as extend thedistribution range of I. crenata to Micronesia. These arethe first records of the genus Incendia for the TropicalNorthwestern Pacific marine province (Spalding et al.2007).
MATERIALS AND METHODS
Collection and morpho-anatomical analysis
Samples were collected by reef wading, snorkeling, and diving at various sites across five islands in Micronesia: Guam, Tinian, Chuuk, Pohnpei, and Kosrae (Fig. 1). Samples were preserved in silica gel and air-dried before being deposited at the University of Guam Herbarium (herbarium code GUAM). Anatomical observations were conducted using both scanning electron (SEM) and light (LM) microscopy. Material used for anatomical observations was hand-sectioned. Prior to sectioning, material used in LM observation was first decalcified in 0.08 M HCl. Some samples were dyed using aniline blue, while others were not dyed prior to setting them atop glass slides. Anatomical observations were conducted and photographed using a Nikon Eclipse 80i light microscope (Nikon Corporation, Shinagawa, Tokyo, Japan) or a Swift SW380T Trinocular Compound Microscope (Speed Fair Co., Ltd., Hong Kong, China). For SEM observations, material was not decalcified prior to sectioning. Sections were embedded on 12.7 mm pin mounts and sputter-coated with gold to a maximum thickness of 5 nm (~15 mA, 120 seconds) using a Luxor Goldcoater sputter coater (NanoScience Instruments, Phoenix, AZ, USA). Anatomical observations were conducted and photographed using a Phenom XL G2 desktop scanning electron microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA) operating at 10 kV.
Molecular analysis
Total genomic DNA was extracted following the protocol described by Saunders and McDevit (2012) or using the GenCatch Blood & Tissue Genomic Mini Prep Kit (Epoch Life Science, Inc., Missouri City, TX, USA) following the manufacturers’ bench protocol. Polymerase chain reaction (PCR) was used to amplify COI-5P using the forward primer TS_COI_F01_10 (Mills and Schils 2021) and the reverse primer GWSRx (Saunders and McDevit 2012) according to the profile reported by Mills and Schils (2021). The 3′ end (~950 bp) of rbcL was amplified in two overlapping parts using the primers F492-R1150 and F993-RrbcS Start (Freshwater and Rueness 1994) following the profile reported by Kucera and Saunders (2012). All PCR products were sent to Macrogen Inc. (Seoul, Korea) for DNA sequencing.
Individual COI-5P and rbcL alignments were generated using the MUSCLE plugin (Edgar 2004) in Geneious Prime 2023.2.1. (Kearse et al. 2012). The optimal evolutionary models for both alignments were selected using jModeltest 2.1.3 (Darriba et al. 2012). Sequences generated during this study were aligned with those from related genera and other Incendia species from GenBank and Barcode of Life Database (BOLD) (Supplementary Table S1). The sets of 42 COI-5P sequences and 23 rbcL sequences were aligned for phylogenetic and species delimitation analyses. Genetic distances were computed as p-distances calculated in Geneious Prime.
Phylogenetic analyses were performed for all alignments using maximum likelihood (ML) methods in RAxML (Stamatakis et al. 2008), and node support was estimated using nonparametric bootstrapping (1,000 replicates). Bayesian inference was also completed using the MrBayes (Ronquist and Huelsenbeck 2003) plugin in Geneious Prime, where each alignment was run for 1,000,000 generations with trees sampled every 100 generations, and the first 3,000 trees discarded as burn-in. The average standard deviation of split frequencies reached less than 0.01 for all Bayesian trees. The automatic barcode gap discovery (ABGD) (Puillandre et al. 2012), assemble species by automatic partitioning (ASAP) (Puillandre et al. 2020), and Poisson tree processes (PTP) (Zhang et al. 2013) methods were used to differentiate and partition Incendia sequences according to phylogenetic species hypotheses. PTP, which identifies putative species using a given phylogenetic tree as an input, was performed via web server (https://species.h-its.org/ptp/) (Zhang et al. 2013) using the ML trees generated for this study as input. PTP analyses were conducted using the following parameters: Tree = rooted, number of Markov chain Monte Carlo (MCMC) generations = 150,000 for COI-5P and 100,000 for rbcL, Thinning = 100, and Burn-in = 0.1. MCMC chains approached convergence for both PTP analyses. ASAP analyses were run with the Kimura 2-parameter (K2P) distance model (Kimura 1980) using the default parameters. ABGD analyses were conducted using the K2P distance model and the following parameters: Pmin = 0.001, Pmax = 0.1, Steps = 10, and X = 1.0. All alignments, evolutionary model selection outputs, trees (ML and Bayesian), distance matrices, and species delimitation outputs generated and analyzed for this study were deposited in Zenodo (European Organization for Nuclear Research, Geneva, Switzerland), a publicly accessible research repository (Mills and Schils 2025; https://doi.org/10.5281/zenodo.17823807). These were used in conjunction with anatomical observations to describe new species and provide a new species record of Incendia crenata for Micronesia.
RESULTS
Molecular analyses
A total of 29 new and 1 previously published (Mills et al. 2022) sequences were obtained for Incendia species from Micronesia, among which were 18 COI-5P and 12 rbcL sequences. Phylogenetic analyses of COI-5P supported the occurrence of I. crenata and the recognition of eight new Incendia species from Micronesia (Fig. 2). Both ABGD and PTP species delimitation analyses also supported the recognition of the eight proposed new species (Fig. 2). The best supported ASAP output was largely congruent with the ABGD and PTP results. However, the ASAP result was less conservative, as it resolved I. crenata as four separate species, and it split I. micronesica sp. nov. specimens from Chuuk and Tinian into two separate species (Fig. 2). The topology of the rbcL trees supported the phylogenetic separation of the six new Incendia species for which rbcL sequences were obtained (Fig. 3). Species delimitation analyses of the rbcL alignment were largely incongruent and failed to reliably resolve species boundaries (Mills and Schils 2025).
There was an average of 9.9% COI-5P sequence distance between Incendia species, ranging from 8.0% (I. gordonii sp. nov.) to 12.1% (I. tsudae sp. nov.). When compared to congeners based on COI-5P sequence data, the new Incendia species differed by 8.1% (I. chamoruensis sp. nov.), 10.0% (I. dixonii sp. nov.), 10.3% (I. fragilis sp. nov.), 8.0% (I. gordonii sp. nov.), 10.4% (I. lobbanii sp. nov.), 8.5% (I. micronesica sp. nov.), 10.5% (I. orotensis sp. nov.), and 12.1% (I. tsudae sp. nov.) on average. Despite a relatively low minimum interspecific distance of 3.4%, phylogenetic and species delimitation analyses recognized I. chamoruensis sp. nov. and I. micronesica sp. nov. as closely related sister species. All other new species were separated from their nearest neighbors by more than 4.1% COI-5P distance (Table 1). For species with more than one representative sequence, the maximum intraspecific COI-5P distance was 0.33, 2.25, 0, 0.15, and 2.13% for I. chamoruensis sp. nov., I. crenata, I.orotensis sp. nov., I. gordonii sp. nov., and I. micronesicasp. nov., respectively (Table 1). The six species with rbcLsequences exhibited interspecific distances of 0.8–12.3%(I. chamoruensis sp. nov.), 3.4–12.6% (I. dixonii sp. nov.),1.9–11.9% (I. gordonii sp. nov.), 4.2–11.5% (I. lobbanii sp. nov.), 0.8–12.3% (I. micronesica sp. nov.), and 9.0–11.0% (I.tsudae sp. nov.). For species with more than one representative sequence, the intraspecific rbcL distance was0.15, 0, and 0–0.29% for I. chamoruensis sp. nov., I. gordonii sp. nov., and I. micronesica sp. nov., respectively.
Morphological observations
Incendia crenata K. R. Dixon (Fig. 4A–E)
Distribution and habitat
Previously known only from the type locality Hideaway Island, Vanuatu (17.698° S, 168.268° E). Its distribution range has now been extended to Kosrae and Pohnpei, Federated States of Micronesia.
Specimens examined
GUAM–GH0013505. Federated States of Micronesia. Pohnpei, Bridge between two reefs, 6.896° N, 158.094° E, 15 m depth, Aug 3, 2012, coll. by T. Schils & A. Simeon; GUAM–GH0013604. Federated States of Micronesia. Kosrae, Buoy 53, 5.339° N, 163.034° E, 15–20 m depth, Aug 8, 2012, coll. by T. Schils & A. Simeon.
Description
Similar to the holotype, plants are red to rusty orange with occasional faint concentric bands, firmly attached proximally, and free of substratum at the broad crenate margins (Fig. 4A & B). Crusts are thin, with individual blades 50–140 μm thick. Hypothallial filaments are composed of parallel rows of irregularly shaped to semi-rectangular cells 13–22 μm wide by 7–13 μm tall that give rise to assurgent perithallial filaments at approximately 45° angles (ranging from 40–46°) from their dorsal surfaces (Fig. 4C). Cells in the lower and mid perithallus are irregularly shaped, laterally compressed (14–26 μm tall by 3–9 μm wide), and have frequent radial cellular projections that are often secondarily pit-connected to cells of adjacent filaments (Fig. 4C & D). Cells in perithallial filaments decrease in height dorsally, such that those in the upper perithallus are approximately isodiametric (3–7 μm tall by 3–7 μm wide). Bullet-shaped hair cells or trichocytes (7–14 μm tall, 5–10 μm wide) borne on short filaments are often observed embedded in the upper perithallus (Fig. 4D). Plants are anchored to the substrate by frequent, relatively long, 6–11 μm diameter, multicellular rhizoids embedded in the thin (11–16 μm) hypobasal cuticle (Fig. 4E). Unlike specimens examined from Vanuatu, reproduction is not observed in specimens from Micronesia.
Remarks
Micronesian specimens of Incendia crenata exhibit vegetative features closely resembling those of the holotype and other specimens examined from Vanuatu. They also exhibit radially directed cellular projections in the lower and mid perithallus, a feature distinguishing I. crenata from the other two species that originally comprised the ‘Incendia crenata species complex’ (I. undulata and I. basilii), as well as the five additional species that have since been added to the complex (I. homosorora, I. chamoruensis sp. nov., I. dixonii sp. nov., I. gordonii sp. nov., and I. micronesica sp. nov.). There are, however, slight differences in vegetative anatomy between specimens from the two regions. Micronesian specimens are notably thinner and have fewer hair cells or trichocytes compared to those from Vanuatu (Dixon and Saunders 2013). The observed morphological differences may be attributed to environmental differences, as well as genetic differences, as evidenced by the maximum intraspecific COI-5P distance of 2.25% between samples from the two regions. Despite the relatively high intraspecific divergence among individuals of this species, phylogenetic and species delimitation analyses strongly support the recognition of these specimens as I. crenata (Fig. 2), hereby extending its known distribution range to include Micronesia.
Incendia chamoruensis M. Mills et Schils sp. nov. (Fig. 5A–E)
Holotype
GUAM–GH0015616. Mariana Islands. Guam, Ritidian Reef, 13.656° N, 144.857° E, 3.5 m depth, May 31, 2019, coll. by T. Schils & M. Deinhart.
Other specimen examined
GUAM–GH0013022. Mariana Islands. Guam, Jannam, 13.506° N, 144.893° E, 7 m depth, May 2, 2012, coll. by A. Simeon.
Etymology
Named in honor of Guam’s indigenous CHamoru people, the traditional owners and caretakers of the land and reefs where this species occurs.
Description
Crusts are maroon to reddish-orange, prostrate, and tightly adherent to the substrate with often free crenate margins (Fig. 5A & B). They are thin and 48–120 μm. Hypothallial filaments are mostly unbranched, parallel filaments of quasi-rectangular to irregularly shaped cells, 30–47 μm long, and 4–8 μm tall which give rise to rhizoids below and assurgent perithallial filaments at narrow angles of approximately 45–60° above (Fig. 5C). Cells in the lower and mid perithallus are oval-like to irregular, tall (10–18 μm), and slender (3–6 μm), but decrease in height as they approach the thallus surface. Secondary pit connections connect radial or occasionally hook-like cellular projections in the lower and mid perithallus to those of adjacent assurgent filaments (Fig. 5C). Cells in the upper perithallus are 5–9 μm tall, 2–6 μm wide, smaller than those in the lower perithallus, and slightly taller than broad to isodiametric (Fig. 5D). Bullet-shaped hair cells are 10–16 μm tall, 6–9 μm wide, and infrequently embedded in the upper perithallus (Fig. 5D). Plants anchor to substrate by thick (8–14 μm diameter), profusely occurring multicellular rhizoids embedded in the thin (6–13 μm) hypobasal cuticle. These rhizoids are significantly longer than the thickness of the crusts and often form complex ‘thickets’ that protrude from the substrate (Fig. 5E). Reproduction not observed.
Remarks
Incendia chamoruensis possesses vegetative features typical of the genus, such as the presence of secondary pit connections in the lower and mid perithallus and attachment by multicellular rhizoids. With the exception of I. micronesica sp. nov. (described below), I. chamoruensis can be separated from other members of the genus by the significant amount of long, thick multicellular rhizoids and the complex ‘thickets’ they form. Additionally, it can be distinguished from the former by the infrequent occurrence of hair cells, the presence of hook-like cellular projections, secondary pit connections in the mid-perithallus, variable angles of assurgent perithallial filaments, and the shape of cells in the upper perithallus. Species delimitation and phylogenetic analyses also support the recognition of I. chamoruensis as a new species (Figs 2 & 3).
Incendia dixonii M. Mills et Schils sp. nov. (Fig. 6A–F)
Holotype
GUAM–GH0013390. Federated States of Micronesia. Chuuk, Etten Island, Northeast Pass, 7.519° N, 151.972° E, 12 m depth, Jul 30, 2012, coll. by T. Schils & A. Simeon.
Etymology
Named in honor of Dr. Kyatt Dixon, who described the genus Incendia, in recognition of his significant contributions to peyssonnelioid algal systematics.
Description
Crust deep red to maroon, with occasional raised patches of orange (Fig. 6A & B). Thallus is firmly attached proximally but forms relatively large, thin blades at the margins (Fig. 6A). Crust relatively thin, between 65–120 μm. Monostromatic, parallel hypothallus composed of irregularly rectilinear cells (8–13 μm wide and 5–9 μm tall) give rise to multicellular rhizoids from their ventral surfaces and assurgent perithallial filaments dorsally. Multicellular rhizoids embedded in the thin 5–7 μm hypobasal cuticle are abundant, relatively thick (6–9 μm diameter), and often extend beyond the bottom of the substrate layer (Fig. 6C). Assurgent perithallial filaments arise at variable narrow angles (25–50°) with cellular projections in the lower and mid perithallus that are oriented radially to approximately parallel to the hypothallus and connect to adjacent filaments via secondary pit connections (Fig. 6D & E). The orientation of the parallel cellular projections occasionally distorts the projection of the assurgent cells in the lower perithallus (Fig. 6D). Cells in the lower perithallus are elongate and laterally compressed (15–22 μm tall by 3–7 μm wide), but decrease in height towards the surface until filaments terminate in cells that are slightly wider than tall (4–7 μm wide and 2–4 μm tall) (Fig. 6F). Hair cells are rarely observed and only differ slightly from other cells in the upper perithallus. Hair cells bullet-shaped, 5–8 μm tall and 4–6 μm wide (Fig. 6F). Reproduction not observed.
Remarks
The vegetative anatomy of Incendia dixonii is markedly similar to the other species within the ‘Incendia crenata species complex.’ However, I. dixonii can be separated from these and other recognized members of the genus by the orientation of the cellular projections in the lower and mid perithallus and the narrow angles (as low as 25°) at which assurgent perithallial filaments arise from the hypothallus. These anatomical differences, in conjunction with phylogenetic and species delimitation analyses of DNA sequences (Fig. 2), support the recognition of I. dixonii as a new species.
Incendia fragilis M. Mills et Schils sp. nov. (Fig. 7A–G)
Holotype
GUAM–GH0017674. Mariana Islands. Guam, Piti, 13.469° N, 144.687° E, 3 m depth, Mar 18, 2025, coll. by T. Schils.
Description
Thallus is crustose, prostrate, burnt-orange to maroon, and firmly attached throughout most of the crust with occasionally free margins (Fig. 7A & B). Plants grow as thin, brittle crusts ranging from 80–125 μm thick with occasional pockets of superimposed growth, where perithallial filaments grow beyond the superficial layer of the parent crust and go on to form a new thallus (Fig. 7C). Hypothallus is parallel, monostromatic and comprised of irregular cells (11–24 μm tall and 6–19 μm wide) that give rise to perithallial filaments at 60–90° angles (Fig. 7D). Cells in the hypothallus and lower perithallus are deeply pigmented, ranging from burnt-orange to red (Fig. 7D & E). Cellular projections in the lower perithallus are oriented transversely and frequently terminate in secondary pit connections that connect cells of adjacent filaments (Fig. 7E). When present, projections often occur serially such that they appear to form a layer of projected cells in the lower perithallus (Fig. 7E). Perithallial cells are irregular to rectangular, laterally compressed, and 13–25 μm long by 6–15 μm wide. Cells decrease in height such that those nearest to the thallus surface are approximately isodiametric (4–8 μm tall × 4–8 μm wide) (Fig. 7D & E). Hair cells are absent or indistinct. Plants are attached by long (up to 215 μm long, 9–14 μm wide) multicellular rhizoids embedded in the 13–17 μm hypobasal cuticle (Fig. 7F). Tetrasporangial nemathecia form entirely superficial patches that extend 120–140 μm above the perithallus surface (Fig. 7G). Nemathecial paraphyses are formed by 6–8 laterally compressed rectangular cells (3–7 μm wide, 13–21 μm tall) that often do not significantly shorten distally (Fig. 7G). Tetrasporangia are frequently produced and borne from basal cells of paraphyses (Fig. 7G). Gametangia not observed.
Remarks
Incendia fragilis possesses the vegetative anatomy typical of the genus. The occurrence of superimposed growth of crusts is currently shared only with its nearest congener Incendia lobbanii sp. nov. (described below). However, the manifestation of said superimposition differed between the two species. I. fragilis could additionally be distinguished from its nearest congener and other closely related taxa by the direction of cellular projections and the lack of hair cells. While tetrasporangia are frequently produced within nemathecia, all observed tetrasporangia were immature. As a result, it was not possible to determine the morphology of mature tetrasporangia for this species. In addition to differences in vegetative anatomy, DNA barcoding and species delimitation analyses strongly support the phylogenetic placement and distinction of I. fragilis from all other members of the genus (Fig. 2).
Incendia gordonii M. Mills et Schils sp. nov. (Fig. 8A–F)
Holotype
GUAM–GH0015358. Mariana Islands. Guam, Togcha Channel, 13.366° N, 144.773° E, 3–8 m depth, Aug 10, 2018, coll. by T. Schils & M. Deinhart.
Other specimen examined
GUAM–GH0016722. Mariana Islands. Guam, Togcha Channel, 13.366° N, 144.773° E, 7 m depth, Jun 3, 2022, coll. by T. Schils.
Etymology
Named in honor of Gregory D. Gordon in recognition of his pioneering research of CCRA diversity in Guam.
Description
Thalli are prostrate, crustose, red in color, and firmly attached to the substrate but free at the undulating to blade-like margins (Fig. 8A & B). Plants grow as individual blades formed by thin crusts ranging from 35–86 μm. Hypothallus is monostromatic, parallel, and comprised of irregularly rounded to oval-like cells (4–7 μm tall and 12–17 μm wide) that give rise to perithallial filaments at variable angles (35–55°) from the distal surface (Fig. 8C). Cellular projections in the lower perithallus are oriented either radially or irregularly parallel to the hypothallial filament and frequently form secondary pit connections with adjacent cells (Fig. 8C & D). When present, the latter type of projection occurs serially, such that they appear to form a layer of projected cells in the lower perithallus (Fig. 8D). Lower perithallial cells are irregularly rectangular, 7–14 μm tall by 3–7 μm wide, and decrease in height distally, to the extent that cells in the upper perithallus are slightly taller than broad to isodiametric (5–8 μm tall by 4–7 μm wide) (Fig. 8C & D). Hair cells are both infrequent and not easily distinguished from other cells in the upper perithallus, making them difficult to observe in cross sections. However, their presence is indicated when observing the thallus surface (Fig. 8E). Plants are attached by multicellular rhizoids up to 120 μm long and 7–10 μm in diameter embedded in the thin (6–8 μm) hypobasal cuticle (Fig. 8F). Reproduction not observed.
Remarks
Similar to I. dixonii, the vegetative anatomy of Incendia gordonii shows a strong resemblance to species within the ‘Incendia crenata species complex’, particularly I. basilii. Though reproduction was not observed, I. gordonii could be differentiated from these and other closely related taxa by the thin crust, as well as the orientation and frequency of cellular projections. In addition to the slight differences in vegetative anatomy, DNA barcoding and species delimitation analyses strongly support the phylogenetic placement and distinction of I. gordonii from all other members of the genus (Fig. 2).
Incendia lobbanii M. Mills et Schils sp. nov. (Fig. 9A–E)
Holotype
GUAM–GH0015266. Mariana Islands. Guam, Ipan Beach, 13.350° N, 144.772° E, 1–3 m depth, Jun 18, 2018, coll. by T. Schils, M. Deinhart & K. Borja.
Etymology
Named in honor of Dr. Christopher S. Lobban for his contributions to the diversity and taxonomy of algae in Micronesia, and in recognition of him as a valued colleague.
Description
Thallus crustose, deep red, and firmly attached throughout much of the crust with orange-red, crenate or blade-like free margins (Fig. 9A). Individual crusts are thin, ranging from 40–115 μm, with occasional pockets of overgrowth resulting in layered, overlapping thalli (Fig. 9B). When this occurs, some perithallial filaments of the lower crust extend into the overlapping layer, creating an appearance of secondary growth (Fig. 9B). The hypothallus consists of parallel filaments of irregular rounded to irregular rectangular cells (5–10 μm tall by 12–18 μm wide) that give rise to assurgent perithallial filaments at variable angles (45–80°) (Fig. 9C). Assurgent perithallial filaments are predominantly regularly arranged. Cells in the lower perithallus are irregular to quasi-rectangular, 6–15 μm tall by 5–11 μm wide, and occasionally form radially oriented cellular projections that connect adjacent filaments via secondary pit connections (Fig. 9C & D). Cells in the mid and upper perithallus are predominantly rectilinear to isodiametric (4–10 μm tall by 3–7 μm wide). The shape of the cells varies according to the thickness of the crust, where they are often more isodiametric when the crust is thinner and more laterally compressed when the crust is thicker (Fig. 9C & D). Rhizoids are 5–7 μm diameter, short (ca. 55 μm length), multicellular, and embedded in the 6–8 μm thick hypobasal cuticle (Fig. 9E). Bullet-shaped hair cells are 16–23 μm tall, 10–15 μm wide, and occasionally embedded in the upper perithallus (Fig. 9D). Reproduction not observed.
Remarks
Incendia lobbanii possesses vegetative characteristics typical of the genus. While some species have exhibited overgrowth via stacking of blades or lobes, I. lobbanii and its nearest congener I. fragilis are the first Incendia species thus far to demonstrate overgrowth through stacked crust layers that are sometimes interconnected by localized areas of secondary growth. The combination of vegetative features, well-supported phylogenetic position, and species delimitation analyses (Figs 2 & 3) strongly support the inclusion and distinction of I. lobbanii as a new Incendia species.
Incendia micronesica M. Mills et Schils sp. nov. (Fig. 10A–E)
Holotype
GUAM–GH0013387. Federated States of Micronesia. Chuuk, Etten Island, Northeast Pass, 7.519° N, 151.972° E, 7.5 m depth, Jul 30, 2012, coll. by T. Schils & A. Simeon.
Other specimens examined
GUAM–GH0013392, GUAM–GH0013393 & GUAM–GH0013394. Federated States of Micronesia. Chuuk, Etten Island, Northeast Pass, 7.519° N, 151.972° E, 12 m depth, Jul 30, 2012, coll. by T. Schils & A. Simeon; GUAM–GH0003381. Mariana Islands. Tinian, 15.039° N, 145.649° E, 7 m depth, Jun 24, 2008, coll. by T. Schils.
Description
Specimens from Chuuk and Tinian possess two distinct habits but similar vegetative morphologies. Crusts from Chuuk are deep red to burnt orange and tightly adherent to the substrate, but free at the broad crenate margins (Fig. 10A). The specimen from Tinian is crimson with occasional patches of burnt orange, tightly adherent to the substrate, and frequently forms free, thin, overlapping blades (Fig. 10B). Crusts thin, ranging from 40–125 μm. Hypothallus is monostromatic. Hypothallial filaments are parallel and composed primarily of rectilinear or crispate cells (25–35 μm wide and 5–13 μm tall) that give rise to assurgent perithallial filaments at approximately 45° angles (between 42–48°) from their dorsal faces and rhizoids ventrally (Fig. 10C). Lower perithallial cells irregular to quasi-rectangular, 11–22 μm tall by 5–10 μm wide, and form radial cellular projections that are occasionally connected to adjacent perithallial filaments via secondary pit connections (Fig. 10C). Upper perithallial cells are smaller than those in the lower perithallus and are semi-rectangular (slightly wider than tall) to isodiametric, 5–12 μm wide and 3–7 μm tall (Fig. 10C & D). Hair cells are frequent, bullet-shaped, 6–10 μm tall and 4–7 μm wide (Fig. 10D). Crusts adhere to substrate via profuse multicellular rhizoids (5–9 μm diameter) embedded in the thin (5–8 μm) hypobasal cuticle. Similar to its sister species Incendia chamoruensis, rhizoids are long, often much longer than the crust is thick, and form complex ‘thickets’ protruding from the substrate (Fig. 10E). Reproduction not observed.
Remarks
Phylogenetic and species delimitation analyses support the position of I. micronesica as a cryptic sister-species to I. chamoruensis (Fig. 2). Despite specimens from Chuuk and Tinian possessing significant habit differences, specimens from both localities are consolidated into one species based primarily on the similar vegetative anatomies of all specimens paired with the results of species delimitation analyses. Incendia micronesica exhibits vegetative characteristics typical of the genus. Like I. chamoruensis, I. micronesica can be differentiated from other members of the genus by the complex assortment of profuse, long, multicellular rhizoids. However, it is distinguished from I. chamoruensis by the shape of the cells in the upper perithallus, the shape and orientation of hypothallial cells, the occurrence of secondary pit connections being limited to the lower perithallus, the consistent angle of assurgent perithallial filaments, and the frequent occurrence of hair cells. The differences in vegetative anatomy in combination with DNA sequence data distinguish Incendia micronesica from I. chamoruensis and other Incendia species.
Incendia tsudae M. Mills et Schils sp. nov. (Fig. 11A–G)
Holotype
GUAM–GH0015400. Mariana Islands. Guam, Tumon Bay, 13.508° N, 144.793° E, 7 m depth, Sep 25, 2018, coll. by T. Schils & D. Gabriel.
Etymology
Named in honor of the late Dr. Roy T. Tsuda for his substantial contributions to the taxonomy and diversity of macroalgae in Micronesia.
Description
Thallus reddish-orange to crimson, encrusting, and completely adherent to the substratum but occasionally less firmly attached at the margins (Fig. 11A & B). Thickness of the crust is highly variable, ranging from 45–200 μm. Hypothallus composed of parallel filaments of rectilinear to irregularly rounded cells (6–11 μm tall and 12–24 μm wide), with assurgent perithallial filaments arising 70–90° from the hypothallus (Fig. 11C). Cells in the lower and mid perithallus lack cellular projections but are occasionally connected to those of adjacent filaments via secondary pit connections (Fig. 11C). Lower and mid perithallial cells are predominantly rectilinear to quasi-rectangular, 8–19 μm tall by 4–12 μm wide, and occasionally branch into two parallel upper perithallial filaments (Fig. 11C). Cells in the upper perithallus are approximately isodiametric to slightly taller than wide (6–13 μm tall by 3–12 μm wide). Growth of cells approaching the margin occasionally follows a flabellate pattern, where hypothallial cells give rise to superior and inferior filaments or where perithallial cells give rise to dorsally and ventrally directed filaments (Fig. 11D). Plants are attached by infrequent and relatively short multicellular rhizoids (4–7 μm diameter) embedded in the 5–11 μm thick hypobasal cuticle (Fig. 11E). Hair cells absent. Tetrasporangial nemathecia form patches that are entirely superficial and elevated 140–175 μm above the perithallus surface (Fig. 11F & G). Nemathecial cells are slender (typically 2–3 μm in diameter), do not significantly shorten distally, and form simple paraphyses typically 10–12 cells in length (Fig. 11F & G). Basal paraphyseal cells cut off cup-like supporting cells that give rise to cruciate decussate tetrasporangia that are 80–100 μm long and 25–35 μm broad (Fig. 11F & G).
Remarks
Incendia tsudae possesses anatomical characteristics similar to I. lisianskiensis, including the angle of the perithallial filaments and the lack of hair cells, a combination that is found in only a few Incendia species. The lack of hair cells is shared with I. glabra, I. lisianskiensis, I. yoneshigueana, and I. lobbanii, while the perithallial filaments arising at up to 90° angles is only shared with I. lisianskiensis and I. fragilis. The method of attachment of I. tsudae is shared only with I. yoneshigueana, while the lack of cellular projections in the lower and mid perithallus is unique among all recognized Incendia species. The occasional flabellate growth at the margin also differentiates I. tsudae from the rest of the genus, as this feature is more commonly associated with species in the genus Metapeyssonnelia Boudouresque, Coppejans and Marcot-Coq. (1976, p. 288). I. tsudae can be distinguished by the unique vegetative characteristics it possesses as well as the significant COI-5P sequence divergence between I. tsudae and all other members of the genus, which is further supported by phylogenetic and species delimitation analyses (Figs 2 & 3).
Incendia orotensis M. Mills et Schils sp. nov. (Fig. 12A–F)
Holotype
GUAM–GH0017651. Mariana Islands. Guam, Orote Point, 13.447° N, 144.619° E, 30 m depth, Mar 7, 2025, coll. by T. Schils.
Other specimens examined
GUAM–GH0017635. Mariana Islands. Guam, Orote Point near Spanish Steps, 13.443° N, 144.618° E, 79.5 m depth, Mar 7, 2025, coll. by T. Schils & M. Mills; GUAM–GH0017652. Mariana Islands. Guam, Orote Point, 13.447° N, 144.619° E, 30 m depth, Mar 7, 2025, coll. by T. Schils.
Description
Thallus prostrate, crustose, crimson in color with occasional raised patches of burnt-orange, and firmly attached to the substrate but free at the undulating blade-like margins (Fig. 12A & B). Blades are thin, ranging from 60–120 μm. Hypothallus is mostly unbranched and comprised of parallel files of irregularly rectilinear cells (5–9 μm tall and 11–20 μm wide) that give rise to perithallial filaments at variable angles (35–85°) from the distal surface (Fig. 12C). Basal perithallial cells are predominantly oval-like to laterally compressed (12–23 μm tall by 2–6 μm wide), and frequently bifurcate into two files of bullet-shaped cells that decrease in size approaching the thallus surface (Fig. 12C & D). Frequent bifurcations occasionally distort the projection of assurgent filaments and result in a densely packed thallus (Fig. 12C). Short, infrequent cellular projections in the lower perithallus are oriented radially and form secondary pit connections with adjacent cells (Fig. 12E). Cells in the upper perithallus are slightly taller than wide to isodiametric, 3–8 μm tall, and 2–6 μm wide (Fig. 12C & D). Hair cells are indistinguishable or absent. Plants attached by relatively long, frequently occurring multicellular rhizoids (8–12 μm diameter) embedded in the 10–16 μm thick hypobasal cuticle (Fig. 12F). Reproduction not observed.
Remarks
Within Incendia, bifurcating or branching perithallial filaments are currently confined to I. regularis, I. orotensis, I. tsudae, and its closest congener I.glabra. Additionally, the lack of apparent hair cellsalso distinguishes I. orotensis from most species in thegenus. Among Incendia species, only I. orotensis andI.lisianskiensis have been reported from mesophoticdepths. I. orotensis was collected at depths of 30 and79.5 m, whereas I. lisianskiensis was described from aspecimen obtained at 55 m. Apart from those, the overall vegetative anatomy of Incendia orotensis is typicalof the genus. Though reproduction was not observed,I.orotensis could be differentiated from I. glabra and I.regularis by the frequency of bifurcation of perithallialfilaments and the orientation and frequency of cellularprojections. In addition to differences in vegetativeanatomy, DNA barcoding and species delimitationanalyses strongly support the phylogenetic placementand distinction of I. orotensis from all other members ofthe genus (Fig. 2).
DISCUSSION
CCRA have historically been difficult to identify due to their inconspicuous presence in benthic communities, occurrence in cryptic habitats, low morphological complexity, morphological convergence, high cryptic diversity, and phenotypic plasticity influenced by environmental factors (Steneck 1986, Hernández-Kantún et al. 2014, Gabrielson et al. 2018). Thus, molecular analyses have become central to investigations of CCRA taxonomy and phylogenetic relationships (e.g., Manghisi et al. 2019, Jeong et al. 2020, Pestana et al. 2021, Mills et al. 2022). Molecular studies on the Peyssonneliales have shown that the order’s diversity is much greater than previously recognized (e.g., Kato et al. 2009, Dixon and Saunders 2013, Ballantine et al. 2016, Mills and Schils 2021, 2026, Nelson et al. 2022). Similarities in morpho-anatomy and the common absence of reproductive features in Peyssonneliales taxa have hampered accurate taxonomic assessments in the past (Pestana et al. 2020). The underestimation of peyssonnelioid diversity is likely even greater in the tropics. Recent collections of CCRA specimens from shallow-water reefs in Guam have resulted in the discovery of a multitude of putative species new to science (Mills et al. 2022), which included the first records of Ramicrusta and Seiria from Micronesia and the description of several new species from Guam (Mills and Schils 2021, 2026).
Dixon and Saunders (2013) reported intraspecific COI-5P divergence ranging from 0 to 2.0%, with a species delineation threshold around 4.0% COI-5P divergence for Ramicrusta and Incendia. Some subsequent studies have also adopted these divergence values and established COI-5P as the standard marker for distinguishing species boundaries for these genera (e.g., Dixon 2018, Pestana et al. 2020, Sherwood et al. 2021). Six of the eight species described in this study are separated from their nearest congener by more than 4% COI-5P sequence divergence, strongly supporting the recognition of Incendia dixonii, I. fragilis, I. gordonii, I.lobbanii, I. orotensis, and I. tsudae as new species. Thedescriptions of these new species were further corroborated by differences in morpho-anatomical charactersand species delimitation analyses (Fig. 2) (Mills andSchils 2025). The inclusion of the previously unreleasedsequences of I. cryptica, I. homosorora, and I. undulata(Dixon 2018), the generitype I. crenata (Dixon and Saunders 2013), and those generated for this study raisedthe number of species with multiple samples to eight,which allowed for a more thorough analysis of inter- and intraspecific divergence among Incendia species.With the inclusion of these sequences, ABGD and PTPanalyses resolved eight new species and I. crenata fromMicronesia exhibiting 0–2.25% intraspecific variationand 3.4–14.7% interspecific variation among Incendiaspecies. ASAP results were less conservative, resolvingI.micronesica specimens from Chuuk and Tinian astwo separate species and splitting I. crenata specimensfrom Pohnpei, Kosrae, and Vanuatu into four separatespecies (Fig. 2) (Mills and Schils 2025). Additionally,ABGD of this dataset resolved a barcode gap distanceof approximately 2.8%, suggesting that a 3.0% COI-5P sequence divergence could serve as a threshold forspecies delineation for this group. This is supported bya number of other recent studies investigating crypticdiversity and systematics in CCRA and other red algaethat have often reported COI-5P barcode-gaps rangingfrom 2–4% (e.g., Freshwater et al. 2010, Hind and Saunders 2013, Hind et al. 2014, Torrano-Silva et al. 2018, Mills and Schils 2021, Deinhart et al. 2022, Mills et al. 2022).
A majority of Incendia species share similar vegetative anatomies and commonly absent reproductive structures that are reported for many peyssonnelioid taxa. Most Incendia species have been described based on single collections, which is not uncommon in this group of red algae (Dixon and Saunders 2013, Dixon 2018, Pestana et al. 2020, Sherwood et al. 2021). This can limit the identification and comparison of diagnostic features, as well as limit comparisons of genetic variation between and within species. As such, few Incendia species possess individual morphological characteristics that distinguish them from the rest of their congeners. Notable exceptions include the lack of cellular projections and occasional flabellate growth at the margins of I. tsudae (Fig. 11D), as well as the complex thickets of rhizoids produced by I. chamoruensis and I. micronesica (Figs 5E & 10E). There is, however, a selection of comparative features of Incendia species that, when examined collectively, are unique for each recognized species (Pestana et al. 2020). The same is true for the eight species described herein, each of which possesses different permutations of features that differentiate them from all other members of the genus (Table 2, Supplementary Table S2). Additionally, the combined orientation and dimensions of vegetative features differed between all nine Micronesian species (Table 3). While the combinations of these morphological characters help reinforce the designation of all eight new Micronesian species, they are particularly crucial when differentiating I. chamoruensis and I. micronesica. In addition to the 3.43% interspecific COI-5P divergence between the two species, they possess several differing vegetative features including the shape of the cells in the upper perithallus, the shape and orientation of hypothallial cells, the location of secondary pit connections, the orientation of cellular projections, the angle of assurgent perithallial filaments, and the frequency of hair cell occurrence (Tables 2 & 3). The multitude of vegetative differences in conjunction with the COI-5P divergence and species delimitation analyses provide sufficient support for the recognition of I. chamoruensis and I. micronesica as cryptic sister species.
A distinct rbcL barcode gap is currently lacking among Incendia species (Mills and Schils 2025), likely due to the relatively conserved nature of the rbcL gene compared to COI-5P, as well as the limited number of species represented by multiple sequenced specimens. Prior to this study, a majority of Incendia species were described on the basis of their phylogenetic position, COI-5P divergence, and morpho-anatomical characteristics (Dixon and Saunders 2013, Dixon 2018, Sherwood et al. 2021). Apart from I. yoneshigueana (Pestana et al. 2020), rbcL sequences effectively served to supplement the description of a select few species. As a result, the overall lack of available rbcL sequence data likely also contributed to the disagreement of species delimitation analyses and inability to reliably resolve species boundaries. Although interspecific rbcL divergence was relatively low between some Incendia species, this phenomenon has also been observed between several other closely related species of red algae (e.g., Boo and Kim 2020, Min-Khant-Kyaw et al. 2025, Taylor and Saunders 2025). As such, species boundaries were primarily determined by a combination of the more conservative ABGD and PTP results, the well-supported phylogenies, the sufficiently high COI-5P divergence, and the unique combination of comparative morphological features. All of these factors strongly supported the recognition of I. fragilis, I. chamoruensis, I. dixonii, I. gordonii, I. lobbanii, I. micronesica, I. orotensis, and I. tsudae as distinct new species, and confirmed the occurrence of I. crenata in Pohnpei and Kosrae.
Furthermore, growing evidence suggests that the diversity and endemism of CCRA are likely much higher than previously estimated and that the broad geographic distributions reported for many CCRA species warrant re-evaluation (e.g., Gabrielson et al. 2018, Twist et al. 2019, Mills et al. 2022). The peyssonnelioid species confirmed to have broad geographic distributions based on DNA sequence data, such as Olokunia boudouresquei (Yoneshigue) Pestana, Lyra, Cassano and J. M. C. Nunes (2021), Polystrata erupta K. R. Dixon (2018), Ramicrusta fujiiana Pestana, G. N. Santos, V. Cassano and J. M. C. Nunes (2020), and Ramicrusta textilis Pueschel and G. W. Saunders (2009), consistently exhibit low intraspecific divergence despite the broad geographic separation between populations (Pueschel and Saunders 2009, Dixon and Saunders 2013, Dixon 2018, Nieder et al. 2019, Pestana et al. 2020, 2021, Mills and Schils 2021, Mills et al. 2022). The high sequence similarity within species from distant geographical regions further supports the recognition of the six Micronesian Incendia species. It also lends further credence to the distinction of I. chamoruensis and I. micronesica as distinct, geographically isolated sister species, an assertion which is reinforced due to Guam’s unique hydrodynamic properties. Guam is surrounded by various transient eddies that, in conjunction with oceanic currents and the size and location of the island, can both promote self-seeding of marine larvae and act as a genetic barrier preventing larval exchange (Wolanski et al. 2003, Kendall and Poti 2014). This can lead to Guam and the southern Marianas possessing significantly different, genetically distinct populations compared to the rest of Micronesia (Priest et al. 2012). While previous studies examined distribution of fish and invertebrate larvae, macroalgae are likely to be affected by these same constraints as most are poor dispersers (Kinlan and Gaines 2003, Kinlan et al. 2005). Conversely, the three species of Incendia with broad and disjunct geographic distributions (I. crenata, I.micronesica, and I. undulata) each exhibit relatively highmaximum intraspecific COI-5P divergences (2.25, 2.13,and 3.14%, respectively). These exceed the typical intraspecific divergences reported for other peyssonnelioidspecies (Dixon 2018), and ASAP analysis split all threespecies into at least two species each (Fig. 2) (Mills andSchils 2025). Despite their broad distributions and considerable genetic variability among populations, thesetaxa were conservatively delineated as single speciesbased on integrative molecular and morphological evidence.
For example, COI-5P sequences from Incendia crenata specimens collected in Tanna, Vanuatu were identical, whereas the type specimen from Hideaway Island (Vanuatu) and newly reported specimens from Pohnpei and Kosrae showed intraspecific COI-5P divergences ranging from 1.0 to 2.25%. A similar pattern was observed in I. micronesica, where specimens from Chuuk diverged by a maximum of 0.15%, while the specimen from Tinian differed from those in Chuuk by up to 2.13%. Although genetic variation within these species is relatively high, the high similarity in vegetative anatomy both within and among Incendia species complicates determining whether specimens of I. crenata and I. micronesica represent geographically isolated populations of their respective species or several distinct species. In contrast, Incendia undulata from Australia differed from the type specimen from Vanuatu by more than 3% (Dixon 2018), suggesting that they represent two distinct species. All three species delimitation analyses support this split (Fig. 2) (Mills and Schils 2025), though we propose that the formal description of a new species should be based on and supported by comprehensive comparative morphological and genetic analyses.
Eighteen peyssonnelioid specimens collected from five islands across Micronesia displayed anatomical and morphological features typical of the genus Incendia. Consequently, this study presents the first record of Incendia in Micronesia, including a range extension for I.crenata and the description of eight new species basedon a combination of morpho-anatomical observationsand molecular analyses. With the nine species examined in this study, the Tropical Northwestern Pacificnow holds the highest reported species richness of Incendia of all marine provinces worldwide. One of thesespecies, Incendia orotensis, also represents the deepestknown Incendia species to date. Specimens were collected at depths of nearly 80 m and at 30 m, which isalso the largest reported depth range any species inthis genus is known to occur. However, this is likely tochange as more samples are collected from mesophoticdepths (e.g., Sherwood et al. 2021). These findings alignwith those reported by Mills and Schils (2021), addingfurther support to the idea that the occurrence of peyssonnelioid species is likely to be significantly underreported, especially in tropical regions. Moreover, thesignificant ecological impact of the rising prevalence ofPAC on tropical reefs (Dutra et al. 2016, Edmunds et al. 2019, 2023, Stockton and Edmunds 2021) underscoresthe urgent need to enhance our understanding of thediversity and ecology of CCRA.
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