Nuphela troglophila sp. nov., an aerophilous diatom (Bacillariophyta) from the Bossea cave (NW Italy), with notes on its ecology.

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Nupela troglophila sp. nov., an aerophilous diatom (Bacillariophyta) from the

Bossea cave (NW Italy), with notes on its ecology

Elisa F

alasco

*

1

, Francesca B

ona1

, Marco I

saIa1

, Elena P

Iano1

, Carlos E. W

etzel2

,

Lucien H

oFFmann2

& Luc e

ctor2

1_{Department of Life Sciences and Systems Biology (DBIOS), University of Turin, Via Accademia Albertina 13,}

I–10123 Turin, Italy; *Corresponding author e–mail: elisa.falasco@unito.it

2_{Environmental Research and Innovation Department (ERIN), Luxembourg Institute of Science and Technology}

(LIST), Rue du Brill 41, L–4422 Belvaux, Luxembourg

Abstract: Nupela troglophila sp. nov. is described from epilithic and epiphytic samples collected around the

artificial lighting system in the Bossea cave, SW Alps (NW Italy). The morphological features of the new species are described and documented through light microscopy and scanning electron microscopy analysis. A comparison with morphologically similar taxa is provided. Nupela troglophila belongs to the group of the small, elliptical–lanceolate species of the genus Nupela. The key feature that distinguishes it from all the other taxa belonging to this genus is the shape of the distal raphe fissures, always laying on the valvar surface, strongly deflected on the same side, but never reaching the valve mantle. The ecological preferences of the most abundant diatom species are explored through the ecological optimum calculation and the Outlying Mean Index (OMI) analysis. Within the Bossea cave, N. troglophila seems to prefer humid and warm sites. The highest abundance was reached on wet walls characterized by low light conditions. Nupela troglophila was generally associated with Humidophila pyrenaica that showed the same pattern of distribution in the cave and similar relative abundance. On the contrary, N. troglophila showed its lowest densities when Humidophila contenta was abundant. Until now, N. troglophila has been recorded only in the type locality.

Key words: artificial lighting system, autecology, biofilm, diatom flora, Naviculaceae, show cave

I

ntroductIon

The genus Nupela VyVerman et comPère

is

morphologically closely related to the families of the

Brachysiraceae D.G. mann and Diadesmidaceae D.G.

mann, sharing with them features related with i) the

shape of the areolae (transapically elongated) and ii)

their density (few areolae composing each stria), iii)

a ridge or a hyaline area at the junction of valve face

and mantle, and iv) simple or inconspicuous proximal

raphe fissures (VyVerman & comPère 1991). Despite

these common features, the genus Nupela presents

unique characters. Cells of Nupela are generally

solitary and small in size, mainly less than 20 µm.

They can be iso– or heterovalvar with regards to raphe

development (sPaulDInG & eDlunD 2008) showing, in

the first case, two fully developed raphe slits, while

presenting reduced or absent raphe on the other valve

in the second case. Valves of Nupela can be slightly

asymmetric with respect to the longitudinal axis and

consequently, they can show an asymmetric central

area. In connective view, valve faces are generally

flat, but sometimes frustules can appear slightly bent.

The external opening of the areolae, often covered

by hymenes that erode during the sample treatment

(PotaPoVa et al. 2003), are transapically elongated,

while small and rounded or oval in the internal view.

The areolae form striae of variable length and extend

as a single row on the valve mantle (s

IVer

et al. 2007).

Raphe slit can be straight or undulated. In external

view, the terminal fissures are clearly bent on the same

side at both poles and they extend down onto the apical

mantle. Proximal raphe ends are simple and sometimes

slightly bent toward the secondary side of the valve.

In internal view, terminal raphe fissures are straight

and a helictoglossa is present. Proximal raphe ends can

be simple, hooked or T–shaped. Sometimes, proximal

raphe ends can be flanked by depressions and small

areolae visible at light microscopy (LM) as refractive

spots. Until now, a total of 60 species belonging to the

genus Nupela have been described (see W

ojtal

2009

and Table S1 of this paper for a complete checklist).

The generitype species of Nupela, N. giluwensis

VyVerman et comPère, was originally collected and

described from shallow mountain lakes (tarns) on the

Mount Giluwe (Papua New Guinea), characterized by

peaty bottom, low conductivity and moderately low

pH (VyVerman & comPère 1991). The genus Nupela

is highly diversified in the Neotropics (metzeltIn &

lanGe–Bertalot 1998), where the genus is often

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present and abundant on periphytic communities in acid

rivers of the Amazon basin (W

etzel

et al. 2012), but

several taxa have also been described from temperate

zones (P

otaPoVa

et al. 2003) or even from Arctic and

Alpine regions (P

otaPoVa

2014). It has been detected

in lentic (

s

IVer

et al. 2007;

B

aHls

2011; m

ItroFanoVa

& G

enkal

2013) and in lotic freshwater ecosystems

(W

ojtal

2009), as well as in intermittent wet habitats

as Sphagnum bogs (k

ulIkoVskIy

et al. 2009) and wet

walls (r

usHFortH

et al. 1984; P

otaPoVa

2011). The

genus Nupela does not show substrate preferences

and it has been found on rocks, wood and saturated

soils (P

otaPoVa

et al. 2003). In general, it is present

in oligotrophic waters (W

ojtal

2009; m

ItroFanoVa

&

G

enkal

2013), but exceptions occur. This is the case of

Nupela pardinhoensis B

es

, t

orGan

et e

ctor

(in B

es

et

al. 2012), typical of moderate TP concentrations (ca.

0.1 mg.l

–1

_{) (B}

_es

_{et al. 2012), N. carolina P}

_otaPoVa

_et

c

lason

in P

otaPoVa

et al., abundant in rivers with 0.75

mg.l

–1

_{P–TP (P}

_otaPoVa

_{et al. 2003), and N. wellneri}

(l

anGe

–B

ertalot

) l

anGe

–B

ertalot

in r

umrIcH

et al.

and N. lesothensis (s

cHoeman

) l

anGe

–B

ertalot

in

m

oser

et al. generally present in polluted rivers (m

oser

et al. 1998; r

umrIcH

et al. 2000; m

onnIer

et al. 2003).

The genus Nupela does not show a pH preference,

being present both in slightly acidic (P

otaPoVa

2013)

and alkaline waters (B

aHls

2011). Most species are

found in low conductivity waters (P

otaPoVa

et al. 2003;

m

ItroFanoVa

& G

enkal

2013; P

otaPoVa

2013; s

ala

et

al. 2014), apart from N. exotica o. m

onnIer

, l

anGe

–

B

ertalot

et j. B

ertranD

(conductivity=1013 µS.cm

–1

;

m

onnIer

et al. 2003) and N. vitiosa (s

cHImanskI

)

s

IVer

et P.B. H

amIlton

(conductivity= 92–206

µS.cm

–1

_{; s}

_IVer

_{& H}

_amIlton

_{2005). In general, Nupela}

species show low relative abundances (ca. 1% for N.

potapovae B

aHls

; ca. 4% for N. decipiens (r

eImer

)

P

otaPoVa

) (B

aHls

2011; P

otaPoVa

2013), but also

exceptions occur: for instance Nupela carolina reached

20% of relative abundance in Contentnea Creek (North

Carolina) and N. neglecta P

onaDer

, r.l. l

oWe

et

P

otaPoVa

in P

otaPoVa

et al. represented 26.5% of the

community in Coles Brook (New Jersey; P

otaPoVa

et

al. 2003). Recently, W

ojtal

(2009) suggested that light

is not a limiting factor for Nupela populations growth.

Despite this, from a literature review on diatom flora

in caves (covering a temporal range from 1900 up

to date), it is possible to conclude that till now only

one species belonging to Nupela has been collected

in subterranean environments, characterized by dim

light (F

alasco

et al. 2014). Indeed, N. thurstonensis

(k

aczmarska

) k

ulIkoVskIy

, l

anGe

–B

ertalot

et

W

ItkoWskI

was collected from wet walls in Thurston

lava tube, Hawaii (r

usHFortH

et al. 1984). The latter

species was collected both on walls and bryophytes,

both close to the entrance and the exit of the lava tube,

but also near the artificial lighting system at the center

of the tube.

In the framework of the CAVELAB project

“From microclimate to climate change: caves as

laboratories for the study of the effects of temperature

on ecosystems and biodiversity”, we performed the

analysis of the diatom flora colonizing the wet and

artificially illuminated walls of the Bossea cave, SW

Alps (NW Italy). The Bossea cave has an important

value both from the aesthetic and scientific point

of view. It is a dynamic cave and the speleogenetic

process is still in progress. Considering its high

naturalistic value, the cave was equipped of two

scientific stations for biological (opened in 1969) and

physical (opened in 1980) studies (P

eano

& F

IsanottI

1994). The Bossea cave was discovered in 1850 and,

as first in Italy, opened to the public and equipped with

artificial illumination in 1874. Even at a first sight, the

walls in the cave are covered by green–brown patinas,

especially in the areas surrounding artificial lights. The

patinas are patchy and present a brighter color near the

most illuminated part of the walls to become sparser

and thinner moving towards the darkest zones. A very

thick brown biofilm was observed at the “Monache”

wall, a vast stone waterfall, artificially illuminated and

never cleaned from the biofilm since the cave opened.

In all the other parts of the cave, where the patinas

are more evident, a cleaning procedure with bleach

is carried out every year. The aim of this paper is to

illustrate and formally describe a new Nupela species,

highly frequent and abundant in the Bossea cave. Some

PAR

(µmol.m–2_.s–1₎ Hmean_(%) Tmean _(°C) Distance.entrance (m)

Cave range of variation 0.2–78.6 51.5–98.5 6.5–10.2 10–427

Optimum Diadesmis gallica 13.36 79.39 9.04 91

Optimum Humidophila contenta 19.13 66.71 8.91 256

Optimum Humidophila pyrenaica 4.15 75.69 9.03 205

Optimum Nupela troglophila 3.24 78.96 9.25 176

Table 1. Environmental parameters: range of variation within the Bossea cave and ecological optima for Diadesmis gallica, Humidophila

contenta, H. pyrenaica and Nupela troglophila in terms of light intensity (PAR), air humidity (Hmean), temperature (Tmean) and distance from

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first indications on its ecological requirements and

distribution within the cave are provided.

M

aterIaland

M

ethods

Study area. Bossea cave is part of the Site of Community

Importance (SCI) IT1160026 (Faggete di Pamparato, Tana del Forno, Grotta delle Turbiglie e Grotte di Bossea) under the managing authority of the Marguareis Natural Park at Frabosa Soprana (836 m a.s.l.) (NW Italy). Bossea cave (44°14'31"N, 07°50'27"E, Corsaglia Valley near Frabosa Soprana, Piemonte region, Italy) is 2800 m long and is structured into two levels (for a total difference in height of 200 m). The lowest part goes from the entrance to the Ernestina Lake. It is characterized by a first corridor (ca. 100 m long), used as location for contemporary art expositions, followed by a succession of big rooms and walls with magnificent speleothems; a river is flowing at the bottom. This touristic part of the cave is artificially illuminated during the visits. The visitors’ pathway ends with a waterfall flowing out from a small tunnel in the wall. The second part of the Bossea cave is not illuminated and scarcely frequented; it develops horizontally behind the waterfall and consists of a series of canyons carved by the river.

Sampling design. We randomly identified sampling sites

distributed all along the touristic pathway, and characterized by the presence of incandescent light. The lighting system is manually regulated and switched on only during the visits. A preliminary mapping of the primary productivity was performed by means of the bbe BenthoTorch® fluorometer probe, developed by ‘bbe Moldaenke GmbH’ for lithic substrates (Schwentinental, Germany). This instrument quantifies and classifies algal chlorophyll–a through the analysis of red light fluorescence emitted from the excitation at different wavelengths. In this way, the bbe BenthoTorch provided chlorophyll–a concentrations of the three main photosynthetic groups colonizing the walls: diatoms, cyanobacteria and green algae. Three replicates per each site were performed. For diatom taxonomical analyses we selected only those sites in which the diatom chlorophyll–a concentration reached at least 0.5 µg.cm–2_{. Following these}

criteria, 31 diatom samples were collected and treated following the standard procedure (UNI EN 13946:2005).

Epilithic, and in some cases, epiphytic diatom samplings were performed by means of a toothbrush and preserved with ethanol (final concentration 50%). Parts of the samples were cleaned from ethanol with following centrifugations. Afterwards, the samples were digested by adding 37% H2O2

on a heater (ca. 80 °C for about two hours). The reaction was completed by addition of HCl (1N). Samples were cleaned again with centrifugations and every time rinsed with distilled water. Cleaned diatom material was mounted in Naphrax® for LM analyses.

For scanning electron microscopy (SEM), parts of the oxidized suspensions were filtered and rinsed with additional deionized water through a 3–µm Isopore™ polycarbonate membrane filter (Merck Millipore). Filters were mounted on aluminium stubs and coated with platinum using a BAL–TEC MED 020 Modular High Vacuum Coating System for 30 s at 100 mA. An ultra–high–resolution analytical field emission (FE) scanning electron microscope Hitachi SU–70 (Hitachi High–Technologies Corporation, Tokyo, Japan) operated at 5 kV and 10 mm distance was used for the analysis. SEM images were taken using the lower (SE–L) detector signal. In each sampling site we measured the following environmental parameters: distance from the entrance (m), distance from the light (m), photosynthetically active radiation (PAR; µmol.m–2_.s–1_{) and luminous emittance (LUX; lux).}

PAR was detected by means of a DELTA OHM S.r.l. DO 9721 probe, LUX were detected by means of a photometric probe LP 471 Phot. Temperature (°C) and relative air humidity (%) were monitored by means of dataloggers (portable meters DO9847 Delta OHM S.r.l.), located near each wall point, at least for one week and set to record environmental variations every hour. Dataloggers in sites F17 and F24 were lost, for this reason they were excluded from the statistical analyses.

Autecology. Considering that sampling points are located

inside a single cave, the ecological characterization of the new Nupela species was conducted at microhabitat level, with respect to the following ecological parameters: light (PAR), mean temperature (Tmean), mean humidity (Hmean) and distance from the entrance (Distance.entrance), which are expected to be the main drivers of diatom distributions in caves (HoFFmann 2002; Poulíčková & Hašler 2007; roldán & Hernández–Mariné 2009; czerWIk–marcInkoWska &

Mrozińska 2011).

The autecology of the new Nupela species was analyzed by the mean of two different approaches: the ecological optimum and the Outlying Mean Index analysis

INERTIA OMI% TOL% RTOL% p–value

Diadesmis gallica 2.84 20.4 10.8 68.8 0.26

Humidophila contenta 6.57 14.8 30.0 55.2 0.01*

Humidophila pyrenaica 3.94 4.0 10.4 85.6 0.21

Nupela troglophila 3.27 11.7 22.6 73.1 0.19

Table 2. Niche parameters of selected diatom species in Bossea cave [(Inertia) variance or weighted sum of squared distances to the origin of the environmental axes; (OMI%) percentage of variability of outlying mean index (marginality), or the deviation of a particular species’ distribution from the overall mean habitat conditions (origin of outlying mean index axes), described by the environmental variables; (TOL%) percentage of variability of tolerance index, which is analogous to “niche breadth” or spatial variance of an organism’s “niche” across the measured environmental variables – a function of all sampling sites with which the species is associated; (RTOL%) residual tolerance (%); p– value = frequency based on number of random permutations (out of 9,999) that yielded a higher value than the observed outlying mean index (p < 0.05 indicates a significant influence of the environmental variables for a species)].

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(OMI). The former method measures the optimal conditions for each environmental parameter, while the second method defines the ecological niche occupied by the species. The ecological optimum was calculated for each selected environmental variable in accordance with the weighted average method proposed by BIrks et al. (1990):

where VOp is the optimum value for the parameter p, Vpi is the value of the parameter p at point i and Ai is the relative abundance of the species at point i.

The ecological optimum was calculated not only for the new Nupela species, but also for other three frequent diatom species.

The Outlying Mean Index analysis (OMI) (DoleDec et al. 2000) was used in order to explore the amplitude of the ecological niche of the new Nupela species in relation to the other dominant species of the cave. The OMI analysis is a two– table ordination technique which places the sampling units in a multidimensional space as a function of the environmental parameters. The distribution of species in this hyperspace represents their realized niches and both marginality and tolerance can be evaluated. The marginality corresponds to the distance between the mean habitat conditions used by a species and the mean habitat conditions across the study area, while the tolerance represents the niche breadth, which means the amplitude in the distribution of each species along the sampled environmental gradients.

For each species, the statistical significance of the marginality was tested by a Monte Carlo test with 9999 random permutations. The frequency of random permutation with values greater than the observed marginality was used as an estimated probability of rejecting the null hypothesis that the environmental gradient does not constrain species distribution. The OMI analysis was performed via the function “niche” in the package ade4 (Dray & DuFour 2007) for the R software (r core team 2013), considering the same environmental parameters and species as for the ecological optima.

r

esults and

d

IscussIon

Nupela troglophila F

alasco, c.e. Wetzel et ector

sp. nov. (Figs 1–30)

Description

LM observations (Figs 1–20): Frustules isovalvar.

Valve lanceolate to elliptical–lanceolate, presenting an

oval outline in the smallest individuals. Length 5.0–

13.1 µm, width 3.1–4.6 µm. Poles rounded or showing

slightly protracted apices. Raphe slits straight, distance

between the proximal ends slightly variable within the

same population. At LM, fine structure of the valvar

surface and ornamentations are not detectable.

SEM observations (Figs 21–30): Externally, outer

openings of the areolae generally covered by hymenes,

that, in some cases, can be partially eroded as a

consequence of the treatment process. Proximal raphe

fissures are straight and simple, sometimes slightly

expanded. Distal raphe fissures are strongly deflected

on the same side and terminate on the valve surface,

never reaching the mantle. Internally, striae are

generally short and composed of 3–4 small areolae,

usually rounded or oval. In general, areolae occupy ⅓

of each hemivalve, leaving a wide longitudinal area.

Central striae are slightly radiate, becoming parallel

towards the apices, 42–50 in 10 µm. The pattern of

striation is not constant, and sometimes striae appear

wavy and disorganized in the middle portion of the

valve. Proximal raphe ends are straight, generally

simple, sometimes “T–shaped”. Distance between the

central fissures is generally quite constant within a

population, ranging from 0.75 to 1.69 µm. Distal raphe

CS1 CS2

PAR 0.6570644 0.61645780

Hmean –0.5939306 0.21250915

Tmean –0.2437798 –0.07832491

Distance.entrance 0.3950750 –0.75411196

Table 3. Environmental variables normed scores on the two axes of OMI analysis [(CS1) first axis; (CS2) second axis)].

Figs 1–20. Light microscope images of Nupela troglophila from sample 11Y (Holotype BR–4391) (1–13) and 4C (14–20). Scale bar 10 µm.

VOp = ∑Vp_∑Ai*Ai

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fissures are straight, terminating in a small and linear

helictoglossa.

Etymology: The epithet of the new species refers to

the troglodyte habitat in which it was collected; in

ancient Greek: τρώγλη, troglê = cavern.

Holotype (designated here): BR–4391 (Botanic

Garden Meise, Belgium).

Isotypes (designated here): BM–101 774 (the Natural

History Museum, London), BRM–ZU9/85 (Hustedt

Collection, Bremerhaven, Germany).

Type locality: NW Italy, SW Alps, Piemonte Region,

Cuneo Province, Corsaglia Valley, Frabosa Soprana,

Grotta di Bossea – Bossea cave. 44°14'31"N,

07°50'27"E. Epilithic samples collected on 26

th

November 2012, on the “Monache wall” by Dr. Elisa

Falasco.

Ecology and associated taxa: Nupela troglophila

was collected in different sections of the Bossea cave,

both close to the main entrance (corridor) and in the

deepest sections, on artificially illuminated walls.

Environmental parameters and corresponding relative

abundance of N. troglophila in the whole diatom

communities are shown in Table S2.

Nupela troglophila shows a preference for

wet walls, where a thin biofilm of water moistens the

surface, even if it was also detected on dry sites. It is

mainly epilithic, but it was also recorded on mosses

and ferns in the corridor. The environmental optima for

N. troglophila are shown in Table 1.

The range of variation of environmental

parameters within the Bossea cave overlaps with

the Nupela troglophila ones. The new species is not

limited by light intensity; indeed its optimum shows

low values of PAR and LUX. It does not seem to be

limited by temperature, with an optimum of 9.25 °C

and it does not require high air humidity.

Compared to other studies (F

alasco

et al.

2014) the flora of Bossea is not very rich, but the

species collected from its walls are very peculiar and

interesting. In total, 23 taxa were identified, so as the

corresponding teratological forms, that were observed

and recorded (see Table S3).

Most of the recorded species are considered

typical of subaerial environments and do not present

a limitation in terms of light. The most abundant

and widely distributed species belong to the genus

Humidophila r.l. l

oWe

et al. (l

oWe

et al. 2014). Even

from a first analysis at LM, it appeared clear that co–

occurrence of different taxa existed. In particular, from

the analysis of the relative abundances, it was possible

Figs 21–24. Scanning electron microscope images (21–22 external view; 23–24 internal view) of Nupela troglophila from sample 11Y (Holotype BR–4391). Detail of the valve apices in the external view (22) with distal raphe fissures strongly deflected on the same side and never reaching the mantle. Detail of the proximal raphe fissures in internal view, straight and simple (24). Scale bars 1 µm.

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Figs 25–30. Scanning electron microscope images of Nupela troglophila from sample 32X (25 external view; 26 internal view) and example of teratological forms, respectively from samples 32B, 32X, 4C and 28Y. Scale bars 1 µm.

to observe a similar trend for N. troglophila and

Humidophila pyrenaica (l

anGe

–B

ertalot

et W

erum

)

r.l. l

oWe

et al. in terms of distribution in the cave

and relative abundance within the communities. On

the opposite, the relative abundance of N. troglophila

showed maximum values in those samples in which the

percentage of Humidophila contenta (GrunoW ex Van

Heurck) r.l. loWe et al. was minimal, and vice versa.

These observations were statistically confirmed by the

niche analysis, performed on these three taxa.

The OMI analysis results are reported in

Table 2. The first two axes of the OMI analysis were

selected. They accounted for 96.4% (73.7% for the first

and 22.7% for the second axis) of the total explained

variability. The first axis is negatively correlated with

mean humidity and mean temperature, while it is

positively correlated with light and distance from the

entrance. The second axis is negatively correlated with

the distance from the entrance and positively correlated

with light, while its relationship with mean temperature

and mean humidity is very weak (Table 3 and Fig. 31).

The OMI analysis revealed different levels of niche

overlap/differentiation among the four species. Figure

31 shows that the niche of N. troglophila overlaps with

the niche of H. pyrenaica as both seem to prefer humid

and warm sites with low light intensities. Humidophila

contenta shows opposite ecological preferences in

relation to mean humidity and mean temperature,

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Fig. 31. Projection of environmental variables on the axis of OMI analysis and representation of ecological niches of the four analyzed species .

thus being found in colder and drier sites, but it seems

not to have particular preferences for distance from

the entrance and light intensity. As shown from the

graph in Fig. 31 and in Table 2, H. contenta is the

most tolerant species and it is the only species whose

mean habitat requirements significantly differ from the

mean habitat values of the Bossea cave (Monte Carlo

permutation test: p=0.03). Diadesmis gallica W. s

mItH

is mainly found in sites with high light intensities and

far from the cave entrance, while it shows no particular

preference for temperature and humidity.

The total percentage of teratological forms

is quite high, reaching a peak of 9.9 at the site close

to the river (see sample 2X in Table S2). With the

exception of sample F4, the highest percentages (>2%

of teratological forms) were found in the inner part of

the cave.

Distribution: Until now only found in the type locality

(Bossea cave) in Italy.

Remarks – Several Nupela species present

morphological affinities with N. troglophila. A detailed

morphometric and morphological comparison with the

most similar taxa is shown in Table S4.

Despite the morphological and morphometric

affinities, N. neglecta is distinguishable from N.

troglophila even at LM, since strongly heterovalvar;

indeed raphe slits on the convex valve of N. neglecta

are distinctly shorter than on the concave valve.

On the contrary, N. troglophila is always isovalvar.

Nupela matrioschka k

ulIkoVskIy, lanGe–Bertalot et

W

ItkoWskI

differs from N. troglophila by the valvar

shape (oval and never showing protracted apices) and by

the larger width of the valves (4.3–6.3 vs 3.1–4.6 µm).

Nupela exotica and N. carolina resemble N. troglophila

both for shape and morphometric characters. The

features distinguishing these taxa are clearly detectable

only under SEM: shape of the distal raphe fissures

(with typical Nupela arrangements in Nupela exotica

and N. carolina) and shape of the longitudinal area

(narrow and straight in Nupela exotica and N. carolina,

while large in N. troglophila). Nupela thurstonensis

is very similar to N. troglophila both for ecology

and morphology. Morphologically, both of them are

isovalvar, but N. thurstonensis shows differences with

N. troglophila in terms of striae density [(30)35–45 vs

42–50 in 10 µm] and in the shape of the longitudinal

area, narrow and linear in N. thurstonensis.

c

onclusIons

Nupela troglophila belongs to the group of the small,

elliptical and elliptical–lanceolate species of the

genus Nupela. The main characteristic feature of N.

troglophila is the shape of the distal raphe fissures,

always laying on the valvar surface, strongly deflected

on the same side, but never reaching the valve

mantle. This morphological character distinguishes N.

troglophila from all the other species belonging to the

genus Nupela.

Nupela troglophila could be confused with

N. thurstonensis both for ecology and morphology.

Indeed, N. thurstonensis was found and described

in the Thurston lava tube, Hawaii (rusHFortH et al.

1984), highlighting its affinities for the aerial habitats

and the ability to grow also under natural and artificial

light system.

In this study, we found a rather high percentage

of teratological forms of N. troglophila. Indeed,

diatom teratological forms in unstable microhabitats

are quite common (Falasco et al. 2009). For instance,

rusHFortH et al. (1984) noticed abnormalities in

the horse–shoe area (or “sinus”) in Planothidium

lanceolatum (B

réBisson

ex k

ützInG

) l

anGe

–B

ertalot

collected from walls in lava tubes. The same authors

found some atypical morphological characters in

several populations collected in this same site, if

compared to the classical species descriptions. This

is the case of Rossithidium pusillum (GrunoW)

rounD et BukHtIyaroVa, Encyonema minutum (HIlse

in raBenHorst) D.G. mann, Eunotia praerupta

eHrenBerG, E. tenella (GrunoW) HusteDt, Adlafia

bryophila (j.B. P

etersen

) G

erD

m

oser

, l

anGe

–

B

ertalot

et m

etzeltIn

, and Pinnularia leptosoma

(G

runoW

) c

leVe

. Deformed valves and irregularities

in the distribution of pores and spines were already

observed in Diadesmis gallica and related to unstable

environmental conditions, such as wide temperature

oscillations, moisture and light conditions (lunD 1945,

1946; GranettI 1978).

(8)

The analyses of the samples collected from walls

around artificial lamps in the Bossea cave revealed a

very peculiar flora, with diatom species dominating

the community mostly unknown. Potential new taxa

belonging to the genera Achnanthidium kützInG and

Sellaphora m

erescHkoWsky

were observed during the

sample analysis and they will be described in further

papers. A deeper knowledge of the flora inhabiting this

cave and its colonization pattern will play a key role

for the elaboration of specific management guidelines

and devices.

Acknowledgements

This contribution is part of the CAVELAB project “From microclimate to climate change: caves as laboratories for the study of the effects of temperature on ecosystems and biodiversity”, funded by Compagnia di San Paolo and University of Turin.

r

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Supplementary material

the following supplementary material is available for this article:

Table S1. Ecology and distribution of 14 new Nupela species additional to the list of Wojtal (2009).

Table S2. Environmental parameters and corresponding relative abundance of Nupela troglophila (normal and teratological cells) in the analyzed communities. Table S3. Checklist of the diatom species and corresponding distribution on cave walls in the Bossea cave.

Table S4. Comparison of the small–celled Nupela species. Data on Nupela troglophila are from the present study. Data on Nupela astartiella, N. carolina, N. exotica, N. frezelii, N. matrioschka, N. neglecta, N. pardinhoensis, N. thurstonensis and N. vyvermanii are taken from the original description.

This material is available as part of the online article (http://fottea.czechphycology.cz/contents)

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Table S1. Ecology and distribution of 14 new

Nupela

species additional to the list of

W ojtal (2009). Species Basionym Ecology Distribution Refer ence Nupela acaciensis V ou Illou D et s. s ala Samples with dissolved oxygen 7.4 mg.l –1, conductivity 10.3 µS.cm

–1, pH 5, temperature 21.3 ºC and altitude 370 m a.s.l.

Colombia (Blanca Stream, Acacias, Lla -nos Orientales) s ala et al. 2014 Nupela americana k oc Iolek in k oc Iolek et al. Recorded in lake Oregon (V an Pritten Lake and W aldo Lake) k oc Iolek Nupela catatumbe nsis V ou Illou D et P la ta –D íaz in s ala et al. Samples with dissolved oxygen 5.1–7.6 mg.l –1, conductivity 3.6–748 μS.cm –1, pH 5.2–7.9, tempe rature 21.1–29.1 ºC and altitude 97–575 m a.s.l. Colombia (Catatu mbo, Meta River , Ca -quetá River , Magdalena Alto and Medio River and

Tomo River basins)

s ala et al. 2014 Nupela decipiens (r eI mer ) P ot a -Po Va Achnanthes decipiens reI mer Low conductivity (19 μS.cm −1) and slightly acid soft water (pH = 6.5) USA (North–Central Pennsylvania, Ge -or

gia, South Carolina)

P ot a Po Nupela elegantula P ot a Po Va Aerial species (moss associated) collected from the wet walls with low conductivity (72–84 μS.cm −1) and slightly acid to circumneutral pH (6.4–6.8) Only known from two localities in Sul -livan County , Pennsylvania P ot a Po Nupela exigua e. t H omas et k o -c Iolek in k oc Iolek et al. Recorded in lake Oregon (Big Lake). Known only from

the type locality

k oc Iolek Nupela fr ezelii P ot a Po Va Its type locality is an urban, at least moderately polluted river

Several locations across the USA

P ot a Po Nupela monoraphia k oc Iolek in k oc Iolek et al. Recorded in lake Oregon (V an Pritten Lake). Known only

from the type locality

k oc Iolek Nupela par dinhoensis B es , t or -G an et e ct or in B es et al. Rivers with eleva ted dissolved oxygen (>7.1 mg L –1), low biochemical oxygen demand (BOD5<3.1 mg L –1) and elevat

-ed total phosphate concentration (0.1 mg L

–1)

Brazil (Rio Grande do Sul)

B

es

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Nupela pennsylva nica ( r .m . P a -tr Ick ) P ot a Po Va Navicula pennsylva -nica r .m . P atr Ick in P atr Ick & r eI mer

Recorded in swamp, springs and lakes

USA (Pennsylvania). This species was usually recorded as A. imperfecta in Eu

-rope and USA

P ot a Po Va 201 1 Nupela poconoensis ( r .m . P a -tr Ick ) P ot a Po Va Navicula poconoensis r .m . P atr Ick

Aerophytic; found on wet walls and swamps

In North America it is not yet recorded outside the Appalachian region. But it seems not to be restricted to North Amer -ica P ot a Po Va 201 1 Nupela pocsii B uczkó et W ojt al in B uczkó et al. Romania Retezat Mountains (S. Carpath

-ians), Holocene deposits

B uczkó et al. 2013 Nupela potapovae B a H ls pH 7.34–8.19; conductivity 12–31 μS.cm −1; temperature 8.6–12.0 ºC USA (Rocky Mountains, North America,

Glacier National Park, Montana)

B a H ls 201 1 Nupela subr ostrata ( H uste D t ) P ot a Po Va Achnanthes subr ostra -ta H uste D t Data from H uste D t (1942): pH 6.7; temperature 8.5 ºC

Sweden Lapland, USA

H uste D t 1942, P ot 201 1 Table S1 Cont.

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Table S2. Environmental parameters and corresponding relative abundance of

Nupela

tr

oglophila

(normal and teratological cells) in the analyzed communities.

Sam

-pling site code Distance from the en -trance (m) Di -stance from light (m) PA R (µmol. m–2 .s –1) LUX (lux) chl –a Gr een algae (µg. cm –2) chl –a Cyano -bacteria (µg. cm –2) chl –a Diatoms (µg.cm –2) Tempera -tur e (°C)

mean and range of variation

Moistur

e

(%) mean and range of variation

Substra -te Nupela troglo -phila relative abundance (%) Nupela troglo -phila terato

-logical forms relative abun

-dance (%)

Total terato logical forms relative abun dance (%)

2X 128 0.5 24.8 1548 0.00 2.47 1.06 8.2 (8.1–8.5) 51.5 (49.8– 53.0) epilithic 0.00 0.00 9.90 4X 124 0.4 13.3 940 0.00 0.43 1.19 8.5 (8.4–9.1) 56.4 (54.1– 58.5) epilithic 0.97 0.00 0.00 9X 158 0.5 15.3 1208 2.88 1.40 1.71 9.4 (9.3– 10.0) 59.2 (56.9– 62.3) epilithic 0.00 0.00 3.18 9Y 158 1.4 3.3 167 0.00 0.37 0.72 9.3 (9.2–9.3) 60.7 (58.6– 63.9) epilithic 1.91 0.00 0.48 10X 184 0.4 78.6 871 0.21 0.07 0.65 9.3 (9.2– 10.1) 54.2 (52.6– 55.7) epilithic 0.50 0.00 6.93 11 Y 204 5.7 1.3 69 0.00 2.5 0.72 9.0 (8.9–9.0) 74.9 (74.0– 77.1) epilithic 44.76 0.00 0.95 12X 202 0.9 23.4 1225 4.13 1.68 1.72 8.8 (8.8–8.9) 77.6 (74.6– 81.2) epilithic 0.00 0.92 2.30 19Y 334 3.0 1.0 61 0.00 1.53 1.79 8.8 (8.8–9.0) 84.7 (73.7– 100.0) epilithic 20.80 0.44 1.77 20X 334 1.4 5.6 324 0.00 1.31 1.29 9.1 (9.0–9.1) 56.3 (55.6– 57.3) epilithic 0.00 0.00 0.48 28X 424 1.7 9.4 437 0.00 2.92 1.73 8.5 (8.4–9.1) 55.9 (54.6– 56.8) epilithic 0.45 0.00 2.25 28Y 424 3.3 0.3 15.9 0.00 0.26 0.64 8.4 (8.4–8.5) 77.9 (72.3– 83.1) epilithic 4.20 0.00 1.68 29X 428 3.0 0.4 22 0.15 0.22 1.19 8.8 (8.7–8.9) 70.3 (68.0– 72.2) epilithic 1.44 0.00 3.37 29Y 428 4.9 0.4 1 0.00 0.13 1.63 8.8 (8.8–8.8) 66.1 (62.5– 69.1) epilithic 6.73 0.00 2.88 32X 378 6.1 0.7 38 0.00 8.92 27.93 8.8 (8.8–8.9) 72.7 (70.1– 75.0) epilithic 72.5 0.42 0.42

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32Y 378 6.5 0.7 42 0.00 15.21 322.74 8.8 (8.8–8.9) 72.7 (70.5– 75.0) epilithic 81.89 0.79 32Z 378 5.4 0.4 27 0.00 4.70 16.91 8.8 (8.8–8.9) 72.7 (70.5– 75.0) epilithic 48.04 0.98 F4 10 9.0 0.2 131 1.32 0.60 0.56 6.5 (4.7–7.6) 97.8 (92.5– 101.2) epilithic 20.31 1.56 F5 16 2.57 2.8 136 0.00 1.82 13.62 9.3 (9.1–9.6) 93.1 (85.6– 100.6) epilithic 87.92 1.45 F6 15 0.29 6.5 841 2.12 1.25 1.56 8.5 (7.6–9.0) 77.5 (75.5– 87.0) epiphytic 1.96 0.00 F12 30 2.0 6.1 390 0.00 0.26 0.94 10.2 (10.1– 10.7) 75.8 (72.0– 79.6) epilithic 4.04 0.00 F13 33 1.5 1.3 84 0.00 0.18 1 10.1 (10.0– 10.4) 90.6 (87.0– 100.7) epilithic 9.09 0.00 F14 33 0.1 11.9 1423 7.30 1.48 0.98 10.1 (10.0– 10.6) 80.1 (71.6– 87.3) epilithic 1.48 0.00 F15c1 35 1.5 5.5 311 4.00 2.00 2.00 10.2 (10.1– 10.4) 61.6 (58.7– 64.7) epilithic 67.94 0.96 F15c2 35 1.5 5.5 311 0.00 0.80 9.00 10.2 (10.1– 10.4) 61.6 (58.7– 64.7) epilithic 25.98 0.98 F17 39 0.2 5.8 377 0.00 2.06 3.12 – – epiphytic 0.00 0.00 F22 68 2.0 2.0 109 0.00 5.24 7.68 10.2 (10.1– 10.6) 98.5 (85.1– 102.0) epilithic 75.93 0.93 F23 75 0.2 12.5 471 3.79 3.05 2.00 9.3 (9.2–9.5) 76.6 (68.5– 82.7) epiphytic 65.40 0.95 F24 87 0.5 4.0 251 4.75 4.7 3.36 – – epilithic 0.00 0.00 F25 93 0.5 2.4 175 0.00 2.71 4.00 8.8 (8.7–9.2) 94.1 (89.5– 98.1) epilithic 4.23 0.00 F26 (b) 100 0.3 19.0 788 0.00 0.49 1.65 8.4 (8.3–9.4) 91.5 (84.9– 96.6) epilithic 0.00 0.00 F26 (d) 100 0.3 19.0 788 0.00 0.49 1.65 8.4 (8.3–9.4) 91.5 (84.9– 96.6) epilithic 0.95 0.00 Table S2 Cont.

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2X 4X 9X 9Y 10X 11Y 12X 19Y 20X

Achnanthidium minutissimum

(kützInG) czarneckI

+ + +

Achnanthidium sp. +

Achnanthidium sp. abnormal form +

Amphora pediculus (kützInG) GrunoW + + Cocconeis lineata eHrenBerG +

Diadesmis gallica W. smItH + + + + +

Diadesmis gallica W. smItH abnormal

form + + + + +

Eolimna minima (GrunoW) lanGe–

Bertalot

+ + +

Eucocconeis laevis (ØstruP) lanGe–

Bertalot

+

Eunotia sp.

Fallacia insociabilis (krasske) D.G.

mann

+ +

Fragilaria arcus (eHrenBerG) cleVe + Halamphora normanii (raBenHorst)

leVkoV

Hantzschia sp.

Humidophila contenta (GrunoW) r.l.

loWe et al.

+ + + + + + + + +

loWe et al. abnormal form

+ + +

Humidophila perpusilla (GrunoW)

r.l. loWe et al.

Humidophila pyrenaica (lanGe–Ber

-talot et Werum) r.l. loWe et al.

+ + + + + + + + +

abnormal form

+ + + +

Microcostatus sp. + + + + +

Navicula crassulexigua e. reIcHarDt + Nitzschia debilis (arnott) GrunoW

Nitzschia solgensis cleVe–euler +

Nitzschia sp. +

Nupela troglophila sp. nov. + + + + + +

Nupela troglophila sp. nov. abnormal

form + +

Sellaphora sp. + + + + +

Stauroneis parathermicola lanGe–

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28X 28Y 29X 29Y 32X 32Y 32Z F4c F5d + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Achnanthidium minutissimum (kützInG) czarneckI Achnanthidium sp.

Achnanthidium sp. abnormal form Amphora pediculus (kützInG) GrunoW Cocconeis lineata eHrenBerG Diadesmis gallica W. smItH

form

Bertalot

Eunotia sp.

mann

Fragilaria arcus (eHrenBerG) cleVe Halamphora normanii (raBenHorst)

leVkoV

Hantzschia sp.

loWe et al.

loWe et al. abnormal form Humidophila perpusilla (GrunoW)

r.l. loWe et al.

abnormal form

Microcostatus sp.

Navicula crassulexigua e. reIcHarDt Nitzschia debilis (arnott) GrunoW Nitzschia solgensis cleVe–euler Nitzschia sp.

Nupela troglophila sp. nov.

form

Sellaphora sp.

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F6b F12a F13a F14ab F15c1 F15c2 F17a F22b F23e + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Achnanthidium minutissimum (kützInG) czarneckI Achnanthidium sp.

form

Bertalot

Eunotia sp.

mann

leVkoV

Hantzschia sp.

loWe et al.

r.l. loWe et al.

abnormal form

Microcostatus sp.

form

Sellaphora sp.

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F24 F25d F26d F26b + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Achnanthidium minutissimum (kützInG) czarneckI Achnanthidium sp.

form

Bertalot

Eunotia sp.

mann

leVkoV

Hantzschia sp.

loWe et al.

r.l. loWe et al.

abnormal form

Microcostatus sp.

form

Sellaphora sp.

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Table S4. Comparison of the small–celle d Nupela species. Data on Nupela troglophila

are from the

present study . Data on Nupela astartiella , N. car olina , N. exotica , N. fr ezelii , N. matrioschka , N. neglecta , N. par dinhoensis , N. thursto nensis and N. vyvermanii

are taken from the original description.

Nupela troglo -phila F alasco , c .e . W etzel et e ct or sp. nov .

Nupela astartiella Metzel

t In et l ange – B er talot Nupela carolina Pot a Pov a et c lason in P ot a -Pov a et al. Nupela exotica o. M onn Ier , l ange –B er -talot et J. B er trand Nupela frezelii Pot a Pov a Nupela ma -trioschka K u -l IK ovs KI y , l an -ge –B er talot et W It K o W sKI

Nupela neglecta Ponader

, r .l . l o W e et P ot a Pov a in P ot a Pov a et al. Nupela par -dinhoen -sis B es , t organ et e ct or in B es et al.

Nupela thurstonensis (Kacz

M ars K a ) K ul IK ovs KI y , l ange –B er talot et W It K o W sKI

Nupela vyver manii

g erd M oser , l an ge –B er talot et M etzel t LM Valve shape isovalvar; lan -ceolate to ellipti

-cal–lanceolate, up to oval in the smallest individuals; apices rounded or slightly pro

-tracted

heterovalvar; elliptical to ellipti

-cal–lanceo

-late; apices cuneate to rounded

slightly hetero -valvar; ellipti -cal–lanceolate isovalvar; elliptical–lan

-ceolate; apices acutely round

-ed and often slightly short– protract-ed heterovalvar; lanceolate to linear–ellipti

-cal

isovalvar; broad elliptical; apices never protracted strongly heterovalvar; lanceolate to ellipti

-cal–lanceo

-late; slightly protracted apices

heteroval

-var; lanceo

-late with subrostrate apices

elliptical to el

-liptical–lanceo

-late; apices often slightly protracted heterovalvar; elliptical to elliptical–lan ceolate; apices cuneate or rounded

Length (µm) 5–13.1 10.0–17.0 5.0(9.3)–15.0 8.6–13.3 10.0–29.0 9.7–12.0 9.0–14.0 10.0–15.0 7.0–13.0 12.0–15.0 W idth (µm) 3.1–4.6 4.5–5.4 2.4(3.4)–4.4 3.0–4.1 3.8–6.3 4.3–6.3 3.0–4.0 5.0–6.0 3.2–4.2 4.5–5.0

Central area shape

wide and unre

-gular

small

squared; lyre– shaped

barely develo

-ped or missing

asymmetrical; reaching the valve mar

gin

on one side of the valve small and round, sometimes miss

-ing

small, round or elliptical small and round or missing

small, round to rectangular big on the epivalve (but not reaching the mar

gin);

small on the hypovalve

Striae in 10 µm 42–50 31–33 42–54 40–45 29–33 42–48 40–48 40 (30)35–45 32

(19)

SEM Terminal raphe fissu

-res

strongly deflect

-ed on the same side and termi

-nating on the valve surface, never reaching the mantle

bent on the same side at both poles and extending down onto the apical mantle

Reference this paper m etzelz In & l an G e – B er talot (1998) [Pag. 378 Figs 46–55] P ot a Po Va et al.

(2003) [Pag. 299 Figs 45–62; Pag. 301 Figs 63–69]

m

onn

Ier

et

al. (2003) [Pag. 279 Figs 1–1

1; Pag. 280

Figs 12–17; Pag. 282 Figs 18–24]

P ot a Po Va (201 1) [Pag.

76 Figs 40–54; Pag. 85 Figs 83–90]

k ul Iko V sk Iy et

al. (2009) [Pag. 15 Fig. 1; Pag. 16 Fig. 2]

P ot a Po Va et

al. (2003) [Pags 295– 298 Figs 1–44]

B es et al. (2012) [Pag. 121 Figs 179–201] k ul Iko V sk Iy et

al. (2009) [Pag. 155 Figs 81–83; Pag. 156 Figs 107–1

10; Pag.

157 Fig. 125]

Type locality and ecology

so far

, collected

only in the type locality (Bossea show cave).

W

et

walls character

-ized by warm air temperature and low light inten

-sities. Found both on natural

-ly and artificial

-ly illuminated sites. Santos (São Paulo), Bra

-zil. Effluent from spring. Contentnea Creek,

W

ilson

Country

, North

Carolina (USA). Submer

ged

woody debris. Small rivers with slow flow char

-acterized by high dissolved oxygen and nitrogen concentrations. tropical fish aquarium, Mardié, Loiret, France. Relatively acid water

, with

low

ion content and carbonates, high nitrate levels. Rhode Island. Benthos. Mod

-erately pol -luted river . Polisto–Lovatsky Sphagnum bog, Novgorod, Rus

-sia. Subaerial habitats with low pH (

Sphagnum

),

oligotrophic.

Coles Brook, Ber

gen

Country

,

New Jersey (USA). Epilithon. Low total phosphorous and nitrogen concentra

-tions; low DOC. Pardinho River

,

Brazil. Epi

-lithon; slow moving flow

, high

dissolved oxygen and total phos

-phorous concentra

-tions.

Thurston Lava Tube, Hawaii. Wet walls near cave entrance (naturally illumi

-nated); moist wall near artificial light within the cave.