﻿Amalophyllonmiraculum (Gesneriaceae), an exceptionally small lithophilous new species from the western Andean slopes of Ecuador

﻿Abstract Recent exploratory field expeditions to the western slopes of the Ecuadorian Andes resulted in the discovery of a new species of Amalophyllon (Gesneriaceae). Amalophyllonmiraculum J.L.Clark, sp. nov. is described from two localities in the Centinela region in the Santo Domingo de los Tsáchilas province. The new species is differentiated from congeners by the pendent habit, basal rosette of leaves, leaf blades with deeply serrate margins, and miniature size. Based on IUCN guidelines, a preliminary conservation status is assigned as Critically Endangered (CR).


Introduction
The flowering plant family Gesneriaceae is in the order Lamiales and comprises 3400+ species in 150+ genera (Weber 2004;Weber et al. 2013).The family is divided into three strongly supported monophyletic subfamilies (Ogutcen et al. 2021) and seven tribes (Weber et al. 2013(Weber et al. , 2020)).The majority of New World members are in the subfamily Gesnerioideae and are represented by 1200+ species and 77 genera (Clark et al. 2020;GRC 2024).Amalophyllon Brandegee is classified in the tribe Gesnerieae and subtribe Gloxiniinae (Weber et al. 2013(Weber et al. , 2020)).
Amalophyllon is a genus of terrestrial or lithophytic herbs distributed from Mexico (Chiapas) through Central America to Venezuela, Colombia, Ecuador, and northern Peru.The presence of subrotate to rotate white corollas defines the following three currently recognized genera from the subtribe Gloxiniinae G.Don: Amalophyllon, Niphaea, and Phinaea.Phylogenetic studies based on molecular sequence data (Smith et al. 2004;Roalson et al. 2005;Roalson et al. 2008;Clark et al. 2011) strongly support the independent origins of subrotate to rotate corollas from recent ancestors with corollas that are bilaterally symmetrical and tubular.The similarity of floral structures in these three clades is likely a convergence that is correlated with vibrational or a "buzz" pollination syndrome (Wiehler 2002).
Amalophyllon was initially recognized as a monotypic genus in the Scrophulariaceae (Brandegee 1914).More recently, the name was applied to Gesneriaceae by Boggan et al. (2008) based on phylogenetic studies that strongly supported a clade that included several previously recognized species of Phinaea.Thus, Amalophyllon was expanded by Boggan et al. (2008) to include 13 species.The description of Amalophyllon miraculum increases the total to 14 species of Amalophyllon.There are currently two species of Amalophyllon in Ecuador and the addition of A. miraculum increases the total to three species (Fig. 1).Amalophyllon divaricatum is known from two populations in southern Ecuador (El Oro and Loja) and at least four populations in Peru (Huánuco, Junín, San Martín, Ucayali).Amalophyllon clarkii Boggan & L.E.Skog is endemic to western Ecuador.The type locality of A. clarkii is in Azuay, near the provincial border of Guayas in the Bosque Protector Molleturo Mullopungo (Fig. 1).Additional populations of A. clarkii are in the Ecuadorian provinces of Guayas and Los Ríos (Fig. 1).The genera Amalophyllon, Niphaea, and Phinaea were recently circumscribed based on phylogenetic studies (Smith et al. 2004;Roalson et al. 2005;Roalson et al. 2008;Clark et al. 2011).A summary of morphological differences between Amalophyllon, Niphaea, and Phinaea is provided in Boggan et al. (2008).There are floral characters that differentiate Niphaea, but there are only fruit characters that separate Phinaea from Amalophyllon.In addition, a fourth clade of rotate flowers is represented by "Phinaea" pulchella (Griseb.)C.V.Morton from Cuba (Clark et al. 2011).Amalophyllon differs from Phinaea by fruit characters outlined in Boggan et al. (2008).The capsules of Phinaea are subtended by erect pedicels.In contrast, the pedicel posture in Amalophyllon is usually curved.The seeds in Phinaea are typically sticky and adhere to the fruit valves, whereas the seeds fall freely from the capsules of Amalophyllon.

Materials and methods
Plants were vouchered and photographed during two field expeditions to the western Andes of Ecuador in 2022 (Clark 2022).Specimens were deposited in the following herbaria: Pontificia Universidad Católica del Ecuador (QCA), Marie Selby Botanical Gardens (SEL), United States National Herbarium (US), New York Botanical Garden (NY), and Missouri Botanical Garden (MO).Digital images of live specimens were taken in the field using a Nikon D100 DSLR with a Nikon 105 mm lens and a Nikon SB-29s ring flash.Morphological observations and measurements were made from live collections, alcohol-preserved material, and digital images using the software program ImageJ (Schneider et al. 2012).
We assessed the extinction risk of Amalophyllon miraculum following the IUCN Red List Categories and Criteria and guidelines of the IUCN (2022).We considered observations, collection localities and population estimates from fieldwork.We refrained from calculating extent of occurrence (EOO) because of the limited number of known populations.The area of occupancy (AOO) was calculated using the software program GeoCAT (Bachman et al. 2011) with the default setting of a 4 km 2 grid.to sparsely pubescent, 2-5 mm long; blade membranous, fragile when dried, oblong to lanceolate, 1.5-5.0cm long, 1-2 cm wide, with 6-9 pairs of lateral veins, margins deeply serrate, bright green with dark green venation on adaxial surface, dark purple on abaxial surface, apex acute.Inflorescence reduced to a single axillary flower (without peduncles), usually produced at the apex of leaves or axis of clustered leaves, with 1-3 flowers per axil, inflorescence bracts absent; pedicels slender, curved, 1.5-2.0cm long; calyx lobes 5, uniformly green, subequal, broadly oblong, nearly free, entire, rounded at apex, ca.2.0 mm long × 1.0 mm wide; corolla lobes 5, fused at base for 1-2 mm forming a shallow tube, tube light red, lobes uniformly white, lobes entire, subequal, spreading broadly during anthesis, apices rounded, corolla lobes broadly ovate, ca.2.5 mm long × 2.5 mm wide, glabrous inside and outside; stamens 4, adnate to the base of the corolla tube, filaments yellow, ca.0.5 mm long, glabrous; nectary absent; ovary nearly superior, subglobose, glabrous, ca. 1 mm long and wide, style ca. 2 mm long, curved, glabrous, stigma capitate.Fruits not observed.

Taxonomic treatment
Phenology.Flowering in March and July.Fruits not observed.
Etymology.The specific epithet reflects the extraordinary and unexpected persistence of remnant forest patches of an area broadly defined as "Centinela" (see next section).Dodson and Gentry (1991) popularized this legendary biodiversity hotspot and brought it to prominence when they reported a mass extinction of plant species from this region.Many of the presumed "extinct" species were recently documented, including Gasteranthus extinctus L.E.Skog & L.P.Kvist (Gesneriaceae) (Pitman et al. 2022).Amalophyllon miraculum is sympatric with Gasteranthus extinctus.The presence of several critically endangered species and the recent discovery and description of new species from Centinela represent a miraculous discovery that has shattered a prevailing assumption that the once-thought-lost biodiversity of Centinela had vanished entirely.The heroic efforts of local landowners who maintained small patches of forests (usually surrounding waterfalls) were instrumental in conserving remnant forest fragments.Also crucial are current conservation initiatives by foundations and academic institutions such as the Ecuadorian conservation NGO Fundación de Conservación Jocotoco and the Jardín Botánico Padre Julio Marrero (JBJM) of the Pontificia Universidad Católica del Ecuador in the nearby city of Santo Domingo.
Distribution and preliminary assessment of conservation status.Amalophyllon miraculum has been collected in Ecuador's western Andean slopes in the Santo Domingo de los Tsáchilas province.The only two known subpopulations are in small patches of forest surrounded by large swaths of deforested agricultural landscapes.The forest patch at the Paul Henry farm is approximately 10 hectares and is located in the northernmost part of the Montañas de Ila range in Recinto Milton Murillo.The southern forest patch in the Bosques y Cascadas Las Rocas private reserve is approximately 50 hectares and lies in the intermontane area between the Andean Cordillera and the northern Montañas de Ila (Fig. 1).These patches are approximately 8 km from each other.The current landowners (Paul Henry and Eduardo Díaz C.) are committed to preserving the forest fragments on their property, but broader efforts are urgently needed by governmental and non-governmental agencies to protect these and other nearby forest fragments.The GeoCAT calculated AOO is 8 km 2 .Amalophyllon miraculum is preliminarily assessed as Critically Endangered (CR) based on a limited area of occupancy (IUCN criterion B1 where AOO <10 km 2 ) and the se-verely fragmented forests (B2a) and ongoing decline of the Centinela forests in western Ecuador (B2bi, ii, iii, iv).Intact forests in the Centinela region are mostly reduced to small (<10 hectares) fragments.Extensive deforestation in western Ecuador, especially Centinela, has resulted in an alarming habitat loss.The area was popularized by E.O.Wilson's (1992) term as the 'Centinelan extinction' because of initial reports of wide-scale plant extinctions by Dodson and Gentry (1991).One of the presumed extinctions was Gasteranthus extinctus L.E.Skog & L.P. Kvist (Gesneriaceae), which was recently documented in more than five forest fragments (Pitman et al. 2022).The rediscovery resulted in a re-evaluation of its IUCN assessment from Critically Endangered (CR) to Endangered (EN).We conducted five field expeditions between 2021 and 2023 and located the only two currently known populations of Amalophyllon miraculum, which is sympatric with the more widespread Gasteranthus extinctus.The only documented populations of Amalophyllon miraculum are inside privately protected areas surrounded by agriculture in unprotected parts of the Santo Domingo de los Tsáchilas province.Effective conservation of this and the other endemic species of the Centinela region will require constant vigilance.
Locating current and future populations of Amalophyllon miraculum is a major challenge because of their small size, ephemeral flowers, and camouflaged foliage on wet moss-covered rock faces.For example, authors Fernández and Zapata recently (April 2024) searched forests in the type locality in Paul Henry's farm but did not locate extant populations.Likewise, it will require targeted and careful searching to document and locate this elusive species.
Comments.Most Amalophyllon have leaf margins that are serrate to crenate.Amalophyllon miraculum and A. clarkii (Fig. 2A) are differentiated from other congeners by the presence of deeply serrate to biserrate leaf margins (Figs 2A, 3A).The leaf blades of A. clarkii are broadly ovate and nearly 8 cm long (Fig. 2A).In contrast, the leaves of A. miraculum are never more than 6 cm long (Fig. 2A).The calyx lobes in A. clarkii are elongate and narrow (Fig. 3C) vs. broadly oblong in A. miraculum (Fig. 2C).Both Amalophyllon clarkii and A. miraculum share a lithophytic habit but differ in their habitat and posture.Populations of A. clarkii were observed growing erect on a rock in the understory of a shaded forest without direct moisture.Populations of A. miraculum are pendent and have only been observed on wet rocks in streams or where mist is persistent.It was common to locate populations of 10-20 individuals of A. miraculum on wet areas of rock faces and no populations on adjacent dry areas, even when mosses and ferns were shared between the two microhabitats.The rosette-forming individuals of A. miraculum were often pendent.In contrast, populations of A. clarkii are either rosette-forming or with elongate erect shoots, but usually erect.There are always five corolla lobes in Amalophyllon miraculum.In contrast, the number of corolla lobes in A. clarkii is usually five, but occasionally six (Fig. 3B).
Amalophyllon miraculum and A. clarkii are geographically isolated.Amalophyllon miraculum is a narrow endemic from the northern lowlands of the western Andes of Ecuador in the province of Santo Domingo de los Tsáchilas (Fig. 1).Populations of A. clarkii are mainly from the southern lowlands of western Ecuador (Azuay, Guayas, and Los Ríos).One disjunct population of A. clarkii was reported in Boggan et al. (2008) from a unicate collection by Alexander Hirtz from the northern province of Esmeraldas.The collection by Hirtz (A.Hirtz 3629 -SEL) could not be located or verified and is therefore not included in the distribution map (Fig. 1).

Figure 2 .
Figure 2. Amalophyllon miraculum J.L.Clark A abaxial view of leaf B front view of flower C lateral view of flower D pendent habit featuring rosette of leaves E adaxial view of leaf.(A, E from J.L. Clark et al. 16805; B, C, D from J.L. Clark et al. 16634).Photos by J.L. Clark.

Figure 3 .
Figure 3. Amalophyllon clarkii Boggan & L.E.Skog A abaxial view of leaf B front view of flower C lateral view of flower D erect habit featuring evenly spaced and rosette-forming leaves E adaxial view of leaf.(A-E from J.L. Clark 13101).Photos by J.L. Clark.

Amalophyllon miraculum J.L.Clark, sp. nov.
Similar to Amalophyllon clarkii, differing in larger and broadly ovate leaves in A. clarkii (>8 cm long) vs. smaller elongate to lanceolate leaf blades in A. miraculum (< 6 cm long); calyx lobes elongate in A. clarkii vs. broadly oblong in A. miraculum; and abaxial leaf surface green with purple venation in A. clarkii vs. uniformly dark purple in A. miraculum.
Description.Lithophytic herb with scaly rhizomes; stem short; pendent to horizontal with leaves in a basal rosette.Leaves opposite, subequal; petiole glabrous